**4. Particulate matter dissolution properties and solution percolation through detritus**

The atom arrangement of the dichotomous earth metal oxide morphs is polyhedral, defined by the number faces of the metal (Titanium, 22Ti) or metalloid (Silicon, 14Si) and oxygen (8 O) bonding, and the configuration forms for TiO2 are anatase metastable octahedral (6 Ti, 9 O, 1, Ti, 6, 9) or rutile stable tetrahedral (8 Ti, 6 O) with differences in atom bonding ratios. The crystalline forms of oxides possess some shape asymmetry-related two-dimensional aspect (2-D, *x*, width, *y*, length), an example of which includes rutile titania and α-quartz silica (crystalline SiO2). The solubility of the

transition metal oxides varies over a range, and is measured as absolute dissolution in moles of solute per liter (M) or fractional solubility (Log *S*). For amorphous Silica (aSiO2), Magnesium Oxide (MgO) and Ceria (III; Ce2O3), the solubility amount is 1.6e-03 - 1.6e-06 M (high solubility); the solubility of that of TiO2, CuO and Fe2O3 is 1.0e-09 - 4.0e-12 M (low solubility); and solubility of Al2O3 and Nb2O3 is intermediate at around 1.6e-08 – 2.5e-08 molar [37] as compared to sodium chloride (NaCl) as an example of alkali metal halogen molecule with equivalents with a solubility of around 6.1 molar.

There are certain relationships in the solubility differences between polymorphs, crystalline and amorphous [2] based on dissolution properties of the crystalline (Rutile) and amorphous forms (synthetic) as in Titania particulates; it is a two phase dissolution, early with a steep decrease in rate of dissolution, and less of a decrease in second phase of the dissolving process. The dissolution kinetics of hard particulates are dependent on free energetics (*G*critical, *α*) with a decrease the critical *G* threshold (less negative) that favors the process with a decrease in interfacial energy (*α*<sup>2</sup> ), specific volume (*ω*), and/or increase in temperature (*T*), in addition to solution electrolyte saturation (1 – *σ*). As the degree of electrolyte undersaturation (*σ*) increases, there is lower solution saturation, and the rate of dissolution increases, but the dissolution of all forms of hard nanoparticles, as compared to ionic particulates, is slow, with the rate constant on the order of 6 nmol per m<sup>2</sup> per sec due to the presence of the solute-solvent interaction energy barrier, a.k.a. surface tension, as the primary variable (Appendix IV. End of chapter educational objectives exercise – Particulate matter toxicity). Particle solubility can also be related by its partition coefficient (+Log *P*, Log *P*), and its entropy of defusion as the temperature (*T*) product parameter in the Gibb's law relationship in which fractional solubility (Log *S*) is favored for solutes with a lower melting point (*MP*) [55] as per Gibbs free energetics. Therefore, there is experimentally-determined indifference in solvation rates between amorphous and para-crystalline forms related to solution desaturation, in which case electrolyte saturation favors regrowth and supports the bio-persistence of both synthetic and harder polyhedrally-bonded particulate matter.

There is the Dreiding potential for the van der Waals (vdW) forces between molecules (Evdw), intramolecular electrostatic attractions and repulsions between atoms of a molecule (EQ) and also intramolecular hydrogen bond energies (EHB) in between molecule atoms, which is represented in summation form, although the three functions themselves are non-linear. In the example of aSiO2 and cTiO2, as to the latter two energies (EQ, EHB), the inter-molecular ionic bond breakage energy is 0.9 J <sup>m</sup><sup>2</sup> [1] for the crystalline particulate and is 1 J <sup>m</sup><sup>2</sup> for the amorphous particulate, and is similar for both as it is the aggregate-to-aggregate interaction energy. The intramolecular (internal) oxide bond cleavage energy difference for the crystalline and amorphous particulates is 10 J <sup>m</sup><sup>2</sup> for the crystalline particulate but is 4 J <sup>m</sup><sup>2</sup> for the amorphous particulate, and is 2.5x for the polyhedral bond configuration of the crystalline rutile [1], which has closer transition element *d*-orbital lattice spacing than the anatase form. The surface adsorbate-dipalmitoylphosphatidylcholine (DPPC) interaction vWV energy is 0.2 J <sup>m</sup><sup>2</sup> for amorphous silica and 0.05 J <sup>m</sup><sup>2</sup> ; and is 1/20th of that of the bulk cleavage energy for amorphous SiO2 and 1/200th of the cleavage energy for crystalline TiO2, also as per the decreased bond length polyhedral bond structure.

Variables apply to water diffusion and also to the diffusion of water-dissolved solutes through a matrix, also known as percolation (Appendix IV). The diffusivity of the test substance though the matrix that can be normalized to its free diffusivity

(*D*0), and the experimentally-determined relationship for diffusion is modelable as an exponential decay function with a respective decrease and increase in solution diffusability with effective molecule size (*a*) and matrix pore size (*r*m) [56]. In the modeling of the relationship percolate diffusability through porous media or matrix, there are differences in relationship between percolate (*p*) permeability or diffusability (*D*(*p*)/*D*0) and matrix porosity (φ)-normalized diffusability of water content through the matrix (*D*<sup>s</sup> (θ) / *D*<sup>s</sup> (φ, phi)). There are both lesser and greater slope, linear and non-linear approximation, fits between solution diffusability and matrix volumetric water content (θ range, 0.0–0.5 a.u.) with cross-overs (θX), 0.0– 0.08 (linear fit model), 0.08–0.145 (non-linear fit), 0.145–0.155 (linear fit) and 0.155– 0.195 (non-linear fit) [57]. Thus, the intervals of the quasi-linear and -exponential relationship are modelable by the power law with different base (*x*-axis) and exponent (slope) variables in which percolation (*p*) is related by the fraction of occupied bonds during filtering flow and the irregular or regular lattice pore filter threshold at critical bond occupation probability (*p* - *p*c, Δ *p*) to the power law rule variable (*t* or *q*) [58] in which a decrease or increase in diffusion is modelable with *q* < 1 (infra-linear) or with a *q* > 1 (supra-linear), while an indirect (inverse) linear relationship between pore ionic interaction coordination number (*Z*) and critical percolation threshold limit (*p*c) exists in three-dimensional systems.

### **5. Bioengineered particulates**

The particulates include the nano-sized small molecule amphiphile-coated iron oxides (8.9–16 nm), the size tunable wavelength-emission quantum dots (5–18 nm), i.e. CdSe interior, ZnS shell RGD-Lys, and proteolytically-degradable proteinaggregate suspension xenobiotic forms such as Abraxane (130 nm), or liposomal formulations of the same [59], size range 100–300 nm to 580 nm [60], with sustained drug release kinetics for biomedical applications and in *in vivo* circulation or as lower distribution size fractions (**Table 2**. Bioengineered nanoparticles). Engineered particles have increased surface area with capacity of exterior surface polyvalency by covalent bound formation to free exterior terminal groups such as amine- or carboxylwith 2x exterior terminal groups per *n*th dendrimer generation and potential for conjugation by covalent linkages; and in addition to the Neocarzinostatin poly( styrene-co-maleic acid) conjugate (SMANCS) [78], the soft nanoparticles include dendrimers and poly-(lactic-co-glycolic acid) (PLGA) in monodisperse size distributions within the NP size range [71], and also include the emulsion- and liposomebased that are approved for human use [70]. By covalent linkage of high-MW polyethylene glycol (PEG) for stealth properties, biocompatibility and blood plasma halflife are improved with a shift to either hepatic and/or splenic RES sequestration [8, 9, 70, 91]; and while soft nanoparticle size and exterior percent conjugation can be tuned by divergent synthesis in comparison the non-covalent type by affinity matching for functionalizing of emulsion- or inclusion-type nanoparticle phospholipid head groups, and emulsion polymerization in case of reactive end group types (i.e. cyano-acrylate monomer).

Generation of uniform distributions of hard nanoparticles is by either bottom-up methods such as by chemical reduction and molecular condensation of multi-atom nuclei [6], or by top-down bulk mechanical milling for example [92]. In addition to the growth of polymorph-bonded atoms, further coating of the surface layer (shell) is required for exterior biocompatibility such as for quantum dots [8, 9], iron oxide [7],



**Table2.**

*(zwitterion,*

 *carboxyl or amine, i.e.* 

*NH2-PLGA-NH2;*

 *sm. mol., small molecule*

> *Bioengineered nanoparticles.*

silicon dioxide [87, 88], emulsion and colloid type [88]. The size and shape of hard nanoparticles can be further modified by differing reaction times, concentration, and via thermal decomposition [83] followed by encapsulation of dispersed monomeric particles by surfactant addition and sonication with resultant droplet formation containing magnetite particles (Fe3O4) as an example of methods applicable for high MW PEG-stealthing of simple phospholipid bilayer liposomes with interior hydrophilic contents [70]. Functionalization of nanoparticles is by surface layer and condensation reaction such in synthesis of silane coat-bonded PEG on SiO2 encapsulated Magnetite cores (Fe3O4) [83], or by the ethero-isophilicity of the exterior surface PEG portion of the dialkene (i.e. 15-C) diphosphorylo- group covalently bound to PEG for shift from renal clearance [74, 75] and stealth property to limit opsonization [76]; the interaction with the SWCNT itself is non-covalent with the alkane part molecule with similar apolarity affinity for the SWCNT wall with dimensional aspect and a + Log *P* lipophilicity. The degradation of the covalent linkage is hydrolytic time constant dependent for the dendritic conjugates, it could be that earlier disassociation of the two portions of the similar partition coefficient (*P*) results in SWCNT nanoparticle opsonization and splenic accumulation, and the free alkyl chain phosphorylo-PEG portion could result in delayed sensitivity by B-lymphocyte IgM, IgG or IgA immunoglobulin response. The less biocompatible polymers include the more hydrophobic, and large diameter hexagonal polymers such as 6(PEG4-erythritol) phenyl group type ([82], **Table 2**) that instead have applications in matrix fabrication, and the polyvinyl chloride (PVC) PEG proportioned crystalline-amorphous composites that have lower transition, melting temperatures (Tm, DSC) [93] and are more deformable than PVC with uses in plastics.

### **5.1 Dendrimers**

The sub-classes of dendrimers include amido-amino dendrimers with core ethylene diamine, (EDA) and diaminobutane (DAB) with amine, carboxyl, hydroxyl or polyethylene glycol (PEG) terminal groups, **Table 2**). Naked heavy metal dye-stained PAMAM dendrimers range in between 1.9 (G1) – 9.8 (G8) nanometers, and with DPTA-/DOTA-chelate functionalization range in between 12.7 0.7 and 13 1.4 nm (Gd-G8; Rh-, Gd-G8) with the number of Gd at 350 atoms per dendrimer (Gd-G8). Inaddition to the monodispersity and narrow size distribution as evident with (Nan) phospho-Tungstate staining by catanionic affinity [17], terminal PAMAM group synthesis is an applicable property as it results in polar molecular anisotropy for DNA van der Waals (vdW) ionic affinity; as result, it is neutral surface nucleic acid transfection by electropolation [61]. The naphtha-sulfonate functionalized Lysine amino acidbased G4 dendrimer [62], BHA.Lys15Lys16(NHCOCH2O)1-(3,6-naphth(SO3Na)32 (BHA = benzhydrylamin), MW 16.6 kDa, is an applied gel sol barrier cream with overall neutral surface and low risk for nanotoxicity; and most recent, there is a polyfunctional group inseries dendrimer hydrogel with internal PEG linker (oxyethanen) and hydrolysis-sensitive ester linkage for improved degradability and less toxicity [63].

The surface anionic PAMAM dendrimers are the half-generations (-COO: G1.5, *n* = 16; G3.5, 12.9 kDa, 5.2 nm; G5.5, 52 kDa, 7.9 nm), and by small angle scattering (i.e. SANS) are spherical shape in deionized solution with low polydispersity (PDI); and there are certain surface physical interaction properties that are associated with anionic macromolecule tissue deposition: i) Caveolin-1-associated protein (AP) mediated endocytic uptake component exists at higher concentration (0.13 μM) since there is lung tissue deposition at lower concentration (0.020 μM) for example in the case of AT1-like alveolar cell/macrophage; and ii) inter-epithelial cellular permeability decreases with increasing half-dendrimer generation between 7.9 nm and 9 nm (- FITC-anionic dextran-70 kDa) due to the junctional complex pore size threshold. A cationic or anionic exterior results in interactions with cell surface receptors or channels with varying affinity, and also results in uptake via internalization mechanisms by non-linear rate kinetics at binding, which is evident by the less than expected blood serum half-live (*t*1/2) to plasma/cell surface interaction as in the case of certain dendrimer types. Since an earlier blood *t*1/2 than expected for the mass density size product of a nanoparticle is consistent with rapid non-selective internalization, this pharmacokinetic parameter can be considered an indicator of toxicity potential [64]. In contrast, the benzene disulphonic acid (BDS) dendrimer ionically-neutralized anionic exterior dendrimer (near neutral) has extended blood half-life [65] with enhanced passive permeation accumulation in tumor tissue. Mixed-surface charge type dendrimers with less effective cationic charge per area for higher affinity binding of cell- and pathogen-released nucleic acid and molecule byproducts, which decreases inflammation pathway activation and toxicity by D- and P-associated molecular pathways (AMPs) with maximum potency and efficacy of deactivation effect for G4 50:50 dendrimers with equivalent terminal amine and hydroxyl groups [66].

High-resolution characterization of Gadolinium (Gd3+)-chelated mass-dense dendrimers is by transmission electron (TEM) with a combination of low- and highdose diffraction techniques, annular dark field (ADF) STEM and energy-filtering (EFTEM) [67], for contrasted detection at high resolution of macromolecule mass and the number of heavy atoms as in the example of Gadolinium (Gd3+)-chelated (DTPA5) monodisperse nanoparticles without the need for heavy metal, i.e. Osmium (190 Da) tetraoxide (OsO4) staining for visualization for a distribution with a median frequency of between 25 and 30 individual nanoparticles within a size range of 12.7 0.7 nanometers (nm) for the Gd-generation 8 dendrimer (600 kDa). With neutral surface nanoparticles there is limited toxic potential, and the mechanism for improved efficacy is by the Maeda effect of passively-enhanced accumulation and retention ('EPR') for tumor treatment is based on blood capillary pore size selectivity [78], and further improvements by conjugated exterior ligand targeting of cell surface receptor overexpression or receptor-specific monoclonal recombinant IgG (12.6 nm)-based therapeutics with non-renal, hepatic and/or splenic clearancedependent circulatory half-lives. As far as macromolecular imaging agents of this class for macromolecular imaging by dendritic conjugates are concerned [68], cyclic chelate Gd-DOTA poly-Lysine dendritic architecture conjugate, Gadomer-17 kDa, is a clinical use due to the concern that acyclic chelates such as diethylenetriamene pentaacetate (DTPA) have the potential for toxicity *in vivo* due to free Gd3+ ion release from chelate. As paramagnetic atom (i.e. Gd3+) chelation affinity increases, water relaxation rate decreases per M\*msec, which results in lower contrast enhancement intensity.

#### **5.2 Micro-emulsion and inclusion emulsion**

Microparticles with insoluble components dispersed in or out of a mixture that scatter light are either: i) colloidal are defined as homogenous non-crystalline matter dispersed into the other but non-sedimentable, and also consist of elements in their unionized element core form in self-association for example in a medium or in deionized water; ii) an emulsion as a colloid with single layer exterior phospholipid

coat (amphiphile); or iii) a suspension with sedimentation possible of one of the substances.

Lipiodol (ethiodized oil) is an ethyl ester of iodized fatty acids with the other lipophilic component a single layered amphiphile stabilizer with 1% of poppy seed oil linoleic and oleic acids, a simple form of emulsion. The toxicity profile of Lipiodol use as radiopaque contrast agent for angiography or lymphography is related to: its rate of infusion and risk for fat emulsion embolism [79], and to it lipophilic constituent composition that results in delayed type hypersensitivity response (Type IV) with repeated administrations. Emulsion or emulsion inclusion type particle size distributions depend on water: oil ratio and are within the millimeter (mm) droplet size range (**Table 2**). The ethiodized oil-in-water (immiscible), or water-in-oil (miscible), emulsion is the two liquid emulsion form that is made a more complex drug-inclusion type emulsion containing less hydrophilic drugs such as Doxorubicin (Daunorubicin, Log *<sup>D</sup>*/vdWD: 0.74 *nm*<sup>1</sup> ) to improve the drug half-life and effectiveness; it is the water-in-oil emulsion that is stable and the improved systemic toxicity profile is by slow continuous infusion of the 62.5% water-in-oil emulsion showing statistical significance by categorical comparison [79]. Doxorubicin (Dox) has greater solubility in Iohexol (75 mg/mL) than in saline (50 mg/mL) than in ethiodized oil; the miscible 1:4 aqueous-to-lipid phase ratio has low stability and disperse distribution of particle sizes; and the release constant (*K*rel; per hr) is most favorable for the 1:4 saline-inlipiodol phase ratio form [80]. The formulation can be made more stable by inclusion of nano-sized hydrophilic NPs such as the PLGA monomers [81] that is tested for its *in vivo* properties such as in the rabbit HCC model in which it is shown that the homogenous intermixed emulsion inclusion Dox form (SSIF) with initial polymer droplet size 100–150 nm (non-covalent, n.c.; **Table 2**) has improved stability by which the Dox (C27H29NO11, MW 544 Da) in PLGA monomer association is more effective than the traditional iodinated formulation (TIF) through blood-tumor barrier (BTB) capillary endothelium fenestrae-interstitium with pore size upper limit 12 nm (un-hydrated particle size/TEM-based; [64]) .

Other emulsion inclusion-based nanostructured lipid carriers also include the PACA sub-class PEBCA (**Table 2**) with more and less lipophilic phases and Cabazitaxel inclusion [77] with low-loading dose renal clearance and high-loading dose (higher MW size) splenic clearance over hepatic due to improved plasma halflife (*t*1/2); and PEG-IR780-C13 uni-micelle (LipImage) has temporal biodistribution kinetics in comparison, first into liver and then to spleen, both tissues being RES cell tissues. The remainder of such soft nanoparticles include the targeted with drug delivery capability based on the poly-lactic glycolic acid (PLGA) repeating unit hydrophile structure and hydrophilicity-based non-covalent polymerization affinity (150–210 nm) [72] with the potential for site bioactivity *in vivo* in monomeric form (1.5–10 nm).

#### **5.3 Silver and gold colloids**

Colloidal particle synthesis is by mixture of an ion solution and hydrogel with applied heat, adsorption upon reduction of oxidation potential, and can be of defined sizes [6]; if uncoated, then surface oxidation results. The colloidal Silver nanoparticles, AgNP20 nm and AgNP70 nm, has been studied in the human eosinophil cell culture model [39], and of the two groups, the smaller NPs with greater surface area-to-volume ratio (AgNP20 nm) result in a wide range of sizes in media solution, i.e. 158.9 nm (μ1) – 1482 nm (μ2) secondary to aggregation by DLS, while the AgNP70 nm

have a lower left-side distribution size (114 nm) and do not aggregate to the same extent due to less oxidizable surface area available for assoc. to the anionic composition of the media. Of these two size distributions of colloidal NPs, only the AgNP-20 nm show an increase in cell apoptosis by Annexin-V staining confirmed by pro-Caspase-3 and -7 decrease, inaddition to Laminin B1 filament protein cleavage; thus, the difference in the potential for cell apoptosis of colloidal elements is related to colloid element oxidation state, and available aggregated colloid anionic surface area for cell surface interaction.

Concerning the size difference between TEM measurement and DLS in the case of the AgNPs, for example the AgNP (NM300K) it can be noted that these are 7.75 2.48 nm (μ σ) in diameter, while size range in solution even after dispersion for this set of colloidal NPs with a much narrower dry distribution (AgNP8 nm) than the AgNP20 nm that aggregate is 28.71 nm or 38.46 nm (μ1, μ2), and 81.37 nm or 97.23 nm (μ3, μ4) in media [39], which varies with concentration (μ1, μ2) and time (μ3, μ4) in solution. For this particle distribution, the aggregation is less skewed the effect remains cell viability decreases with concentration dependence with more of a decrease in viability for the Beas-2B immortalized cell line over the A549 bronchial adenocarcinoma cell line; and due to the poor solubility of AgNP with a less electronegative electrical potential to ionize as per the Pourbaix-pH relationship. Cell viability percent (%) assay, and Comet DNA tail sign assay for chromatin DNA genotoxicity, and cellular oxidative stress-determining assays such as the FPG enzyme assay for oxidized base carbon detection (i.e. oxo-Guanine) or SOD activity and GSH level reduction assays are utilized for determination of such effects [94, 95].

The toxicity of PEGylated colloidal Gold NPs, in the form of spheres or rods, has also been characterized with size and shape determined by TEM, which is similar in length dimension (50 nm, 45 x 10 nm) [10]. The nanospheres are monodisperse with a PDI of 0.02 and with a negative zeta (ζ)-potential (27.1 mV) and agglomerate in solution as per a size of 89 nm by DLS and this *in vivo* would be by association of Citrate impurity, or could be due to association with the R-groups of native albumin (pI = 4.5, anionic), whereas rod-like NPs with aspect will remain near neutral ζ (+1.13 mV). It appears that the presence of surface charge is the primary determinant to particle clearance *in vivo*, which limits tissue interstitial toxicity since the effective exterior anionic spherical particles are hepatic > splenic RES substrates, while the rodlike particles with slight exterior positive charge and 1-D aspect smaller than the tumor tissue pore size accumulate passively in tumor tissue. Based on a recent study of the biodistribution of AgNP or AgNP and AuNP of similar sizes (*log*-normal, μ = 10.82, 10.86, GSM) after co-inhalation exposure to rodents over 28 days [96], at day 1, the AgNP aggregates distribute to the liver and olfactory bulb over the spleen; whereas, with AuNP co-inhalation, the biodistribution of the Silver (Ag, ng/g) is to the spleen over the liver, and is due to the opsonization of the particles, larger effective sizes and retained permeability to the splenic arterial side capillary beds of the red pulp; this is consistent with the more lipophilic and less soluble character of the Gold elemental/ colloid particle that results in its opsonization and splenic accumulation after coinhalation exposure.

#### **5.4 Iron oxide nanoparticles**

The iron oxide NPs (IONPs) have supraparamagnetic water proton-relaxation properties and include the ore-derived Hematite (Fe2O3; FeIII, 2), and also the

chemically-synthesized crystal Magnetite (Fe3O4; FeII, 1: FeIII, 2) form by basic pH ultrasound-assisted co-precipitation of ferric and ferrous chlorides [83], with both iron forms containing unpaired electrons reduced by mono-Oxygen. The various IONPs are (size range): SPIONs (50–180 nm), ultrasmall PIONs (USPIONs, 10– 50 nm) and the very small SPIONs (VS-SPIONs <10 nm) (**Table 2**) The IONPs are *r*2 molar relaxivity contrast agents with above paramagnetic effect relaxational rate properties (*R*2) that result in *T*2-weighted contrast enhancement with percussion frequency around transverse magnetic plane as compared to the Gd3+ and Mn2+ transitional metals with *T*<sup>1</sup> shortening, and longitudinal relaxation rate (*R*1) increasing magnetic properties per concentration of agent per time (*M msec*) <sup>1</sup> with a reciprocal size-dependent enhancement effect high-molar relaxivity (*r* 2 ) paramagnetic effect contrast enhancement for long TR, long TE sequence MRI (T2W-seq) in the inverse correlation IONP size-to-relaxation rate effect [84], and a direct relationship between loading amount and magnetic strength [26]. Silica-coated Magnetite particles also developed with nanoporous silica exterior shell with an aminosilane organic coat for PEGylation and multimodality applicability [7] of either a low or high contrast enhancement molar relaxivity ratio (*r*2/*r*1) [88, 89] (**Table 2**); there also exists a class of cross-linked iron oxides (CLIO) in core for improved stability of exterior surface for amination (CLIO-NH2)n for additional covalency and functionalization [90].

Iron oxide NPs of spherical shape by SEM, and mean size 60 nm by TEM, result in lymphocyte cell viability decrease in an inverse relation to ROS and toxicity upon accumulation, which is responsive to applied Thymoquinone (TQ, *ox*) [95], a Phase II detoxification enzyme (NQO1) substrate that metabolizes to cyclohexa-2,5-diene (methyl, isopropyl)-yl-1,4-diol, with maximum reduction in ROS at mid- concentration in a low, high concentration parabolic relationship of ROS production, and alike to the toxicity type that results in A549 adenocarcinoma cells upon exposure to ZnO NP (NM110) in which oxidative toxicity is more than genotoxicity [94]. Although the potential for interaction with the nuclear DNA exists as determined by UV (200– 350 nm)-visible spectra- scopy, particle toxicity for such less soluble crystalline particulate matter is primary non-genomic extranuclear toxicity. For bioengineered iron oxide NP infusions, the *in vivo* pharmacokinetics and biodistribution is measured by QAR following 59Fe radiolabeled-AMI SPION NP infusion, which results in an increase in transverse tissue relaxivity (*R*2) with T2W-negative contrast enhancement in liver over the spleen or lung organs [97] and is by hepatic macrophagereticuloendothelial system cell internalization.

Endogenous iron in its free oxidized form (Fe3+) readily reduces, and in its hydroxylate ferric form bonds into Apoferritin (18 nm), a variable diameter multi-αhelix 24mer subunit cage (MW 474 kDa) that can hold 3.5–4 Fe (II)/O2 with between 220 and 1900 ( 2220) molecules of <sup>&</sup>gt;50% oxidized ferrous iron (Fe3+) [98] as Ferrihydrite, Fe3+O2H *<sup>n</sup>*H2O [99]. Accrual of iron oxide densities is seen on TEM of pollution-exposed population sample specimens with inelastic scattering EELS spectra showing the iron oxide-containing Hematite, Magnetite and Goethite nanoparticle forms (30–50 nm) [100], some of which, could be Ferritin protein-assoc. forms such as 2 L-Ferrihydrite/ABACA, and Maghemite-like that are neurotoxic and also visible by nano-diffraction TEM of small volume samples [101]. Inaddition to epitheliumand olfactory nerve ending- internalized exogenous particles, the toxicity of the oxide particulates is also from extracellular bioaccumulation potential due to microgliamacrophage overload and deposition as it is in the case of Ferritin adsorption on inosilicates and formed asbestos bodies in tissue interstitium.

#### **5.5 Cesium-, zinc- and cobalt- oxides**

Inaddition to the effects of AgNP-20 nm and AgNP-70 nm particles (Sub-section B), the effects of other major oxide forms of the other transitional metals have also been studied in the human eosinophil cell culture model, and include the rutile and crystalline forms, TiO2 (anatase crystal oxide), CeO2 (crystalline) and ZnO (crystalline) NPs particles have been also studied in the human eosinophil cell culture model [39]. The respective particle aggregate populations follow a multimodal distribution, but could also fit a *log* normalized distribution as a single population per DLS after particles dispersion in cell culture medium such as RPM1 1640 with particle size measured in-solution by dynamic light scattering, and in the examples of CeO2, ZnO and TiO2 in which the *log*-normalized CeO2 particle distribution is left-shifted as compared to for the other two with a smaller particle size distribution; and as to the cell viability and gene expression effects, the elemental oxides with favorable electropotentials to maintain in dry/TEM particle size (TiO2-NM101), or decrease in size upon de-aggregation (CeO2) have less potential for negative effects on cell viability and toxicity [94], and of these three oxides, the effect of the largest dehydrated particulate of the NM series, ZnO-NM110 (132 nm, TEM) is towards ROS generation [94] and apoptosis [39]. The molecular gene expression changes that occur as a result of cell internalization include the activation of IL-8 and MIP-1α/1β (ZnO, AgNP <sup>20</sup> nm), RANTES (ZnO) genes with non-change in *CXCL9* (*alias* MIG) gene and cytokine levels gene expression [39].

The genomic effects following the local intratacheal instillation of Cobalt NP (- Nano-Co, 50 μg/mouse; μ = 20 nm, TEM) and Nano-TiO2 (μ = 28 nm) have been studied in the mutant guanine phosphoribosyltransferase (*gpt*) assay for selection of positive *Cre*-activated cell clones with integrated transfections by application of 6 thioguanine to cells in culture, and crossed transgenic mice study endpoints [40]. Based on such study data, it can be specified that: i) there is an increase in local protein concentration due to increased permeability of capillaries, and decreased neutrophil number is due to cell diapedesis inaddition to an increase in local inflammatory cytokine (*CXCL1*) concentration; ii) there is an increase in genomic DNA base transversions (G- > T) with Nano-Co colloidal Cobalt suspension exposure, but not with control or Nano-TiO2 exposure as determined by 8-OHdG levels and non-WT *gpt* gene single-pair base sequence mutation frequency analysis; and iii) there is cell cycle progression upon exposure to Nano-Co that surface oxidizes (Co2+) with the potential for assoc. anionic serum/plasma constituents, and can be seen in lung parenchymal cells by IHC staining for *PCNA* and Ki67 (*MKI67*) positivity as compared to Titania (TiO2) that is reduced and in oxide form. There is a probability that there is correlation to bond dissolution rates as the water solubility (molmetal/L) of titanium- (Ti2O3, TiO, n/a; TiO2, 10<sup>9</sup> M) and iron- oxides (FeO, Fe2O3, 10<sup>10</sup> – 10<sup>12</sup> M) is low [37], the oxide-types being Ferritin cage substrates; and the potential for inflammatory/ ROS stress, DNA alterations and cell proliferation/transformation exists with high concentration exposure to transition metals of oxides that have higher solubility.

#### **5.6 Titanium oxides**

Cell membrane (CM)-associated cellular esterase-mediated dichlorofluorescein diacetate (H2DCFDA) acetate cleavage and reduction (H2DCF) assay is coupled to the with DCF oxidation fluorescence assay for ROS detection; by the cell culture-based ROS generation assay, amorphous and rutile TiO2 particle matter reactivity is

determined by size and crystal phase [41]. TiO2 samples are prepared by aerosol reactors, particle size distributions differences are generated by spectrometry (SMPS), and morphologic characterization of the size distribution is by filter paper electron microscopy (SEM, TEM) and by x-ray diffraction (XRD) for particle size characterization of particle distribution phases. There are monodisperse distributions of amorphous phase TiO2 particles within the >30 ≤ 53 nm size interval are most reactive in peroxide generation (H2O2) per particle surface area (S.A., μmol/m<sup>2</sup> ). Particle number and concentration are the variables for 3 nm-sized particle-mediated ROS, while 41–53 nm particle size is favorable to cell ROS generation in the order of amorphous > para-crystalline (anatase) > rutile for a 34–102 nm range particle size distribution. In this study design, particle number and concentration are the additional variables to particle size with increased ROS stress determined for the amorphous particles in a single time point experimental data acquired at 15 min. in which case solubility differences would be negligible between the particle types.

The dose-dependent effects of the anatase and crystalline forms of TiO2 particulates are assessed in the 3 T3 fibroblast cell culture model exposure model by nuclear division index (NDI) and the Cytochalasin B F-actin inhibitor binucleate micronucleation chromosomal damage (BNMN) assay with non-aggregated mean particle hydrodynamic size ranges (DLS) determined in cell culture medium for nanosized anatase (An-10; 26 nm), bulk anatase (B-An; 260 nm), nano-sized rutile (Ru-10; 82 nm) and bulk rutile (B-Ru; 755 nm) [42]. There is maintained cell internalization to a minimum of 28 nm (An-10; > 28 ≤ 53 nm) for anatase forms (TiO2) with no change either assay (BNMN, NDI), however this is not the case for the rutile forms, which have a right-shifted bimodal size distribution, aggregate, and are present in the media supernatant rather than within cells at 24 hrs; the B-Ru particles either endocytose less, or endocytose initially. The 82 nm-mean size Ru-10 crystalline particles are more oblong in shape (TEM) with a one-order more negative zeta (ζ)-potential (DLS) than the egg-shaped/spherical An-10 particles.

#### **5.7 Quantum dot nanoparticles**

The intermediate group metal-metalloid QDs with semiconductor properties have been applied for biochemical luminescence detection [85, 86]. The quantum dots are comprised of a transition metal (Group 12; i.e. Cd, Zn) and metalloid (Group 16; Se, S) bonded atoms as core (CdSe) and shell (i.e. ZnS) particles with conduction to valence band orbital fluorescence emission (*E*<sup>m</sup> <sup>λ</sup>), **Table 2**. Being of monodisperse nanometer scale size distributions, the particles have applications in electronics including for LED photoluminescence (PL). QDs with Tungsten Sulfide (WS2) core have a disperse distribution with one modal interval in-between 4 and 6 nm, and absorbance (*λ*abs) wavelengths at 246, 278, 333, 365 nm and *E*<sup>m</sup> <sup>λ</sup> in-between 370 and 500 nm wavelengths with this range being conducive to wide-range of emissions color application. As to the shell and organic coating, there is a concentration-dependent cation interaction-mediated decrease in PL intensity applicable to the detection of a number of milieu transition metals (Pb, Cd, Hg, Fe3+ (Fe2+), Cu) [86], and also in other functionalized organic molecule-coated QDs such as Molybdenum Selenium (MoSe2/COO, NH3+, SH) consistent with ionic chelate neutralization that results in an increase in emission intensity (i.e. Cu2+/COO) [85], which also affords efficiency in detection by threshold-based binomially. PEGylation- and peptide-incorporated quantum dot-based NPs with larger size (> 12 nm) with increased blood circulatory *t*1/ <sup>2</sup> time and Stokes shift emission for PL at λ800 nm (NIR) [8] with applicability for

effect on only irradiated tumor tissue angiogenic capillary-interstitium barrier pore size that can also be studied by other small NPs with monodisperse distributions in the lower nano-size range NPs [102]; and the initial feasibility of targeting to vascular malformations can be studied by endothelium-targeted QDs nanoparticles (i.e. α<sup>V</sup> β<sup>3</sup> integrin, [69]).

The size distributions of Cys-QDs have been shown to be narrow per QD color (*E*<sup>m</sup> <sup>λ</sup>, 515–584 nm) and within the 2.85–4.31 nm range by TEM for dry particle size, and within the 4.36 (GFC), 4.64 nm - 7.22 (GFC), 8.65 nm (DLS) range by size exclusion chromatography and dynamic light scattering in solution {Choi, 2007 #9} [9]. There is a difference in aggregation potential for particles with non-neutral exterior properties, but indifference in size for zwitterion ion organic molecule-coated shell wet QDs inaddition to those in solution with DHLA-PEG coating and near neutral exteriors. Cys-QDs show renal elimination clearance after direct infusion with threshold limits determinable by bladder intravital fluorescence microscopy (IFM) intensity; there is an increase in blood half-life (*t*1/2) beginning at a hydrodynamic diameter (HD) of 4.99 nm [9], which is consistent with the pore size limit to peritubular renal capillary secretion. The pharmacodynamics of tissue distribution for neutral charge QDs with larger diameters (8.65 nm) and zwitterionic surface is hepatic > lung (> spleen), and that of smaller particles (4.36 nm) is a slight shift towards hepatic elimination due to the renal peritubular capillary reabsorption pore size threshold; splenic RES cell clearance is for non- cationic, anionic or -cationoneutral micro-sized particles.

#### **5.8 Pharmacokinetic models and hyper-permeability analyses**

The circulatory transcapillary transfer potentials (*P*c, *P*i, *π*c, *π*i), reflection coefficient (σ) and permeability coefficient (*L*p) are the three variables in the relationship for hydraulic flux calculation; first the forward transfer water flux per unit area (*J*v/A) is determined, and then the ratio of the rate of solute flux-to-rate applied of water flux ratio (*J*s/*J*v; a.u.) is determined [103]. Since hydraulic or water conductivity coefficient (*L*p)-adjusted flux is affected by the presence of circulatory macromolecular particulate matter, there is the Peclet variable adjustment in the modified Starling relationship [104]; Appendix I. Microvascular fluid exchange and pharmacokinetic modeling); and the relationship is also applicable for determining of hydraulic flux of water (or solute in Ref. to water) in the presence of pharmacologically-applied macromolecule interactions in modeling secondary to exterior vdW hydrophilicity interactions of capillary wall permeable macromolecules with water; there are two relationships, one that is of hydrostatic pressure favoring filtration when there is a lower Peclet coefficient, and the other of the effect of the permeable fraction of the macromolecules and increased oncotic effect on water filtration. The overall model for transcapillary permeation is a three layer plus one layer barrier [105] in which there are three capillary layers (EGL, endothelium, basement membrane) with the fourth layer being the interstitial matrix, and at which vdW interactions occur.

Endothelial barrier hyper-permeability and the altered pharmacodynamics of affected tissues can be also be studied *in vivo* by real-time quantitative MRI (qDCE-MRI) at high resolution and analyzed by contrast enhancement-based multivariable compartmental modeling with several parameters, *C*<sup>t</sup> (*t*), *C*<sup>p</sup> (*t*), *V*p, *K*ep and *V*<sup>e</sup> and the time (*t*), time constant (τ)-dependent fractional solutions for the forward transfer constant, *K*trans, are determined (Appendix I. Microvascular fluid exchange and pharmacokinetic modeling). The pharmacokinetic toxicity assessment-applicable models have the common assumption that the transfer between compartments is linear

although plasma concentration-coupled tissue clearance (*K*ep, per min) is exponential decay (1/*e* Kep*t* ) for (small) molecules that are renally-cleared and less toxic. The common relationship for the DCE-MRI pharmacokinetic models are based on the earlier Crone indicator dilution method [106] in which tissue extraction (*E*, 1 – eP\*SA/F; Appendix I) is related to experimentally-determined tissue capillary permeability-surface area product (*P*\**S.A.*, a.k.a. *K*trans) normalized to tissue blood flow (*F*); tissue extraction is also related to clearance (efflux), *e* Kep/F.

There is also a model relationship for the non-linear (integrated) increase in tissue volume of distribution (VD') over the baseline (*V*D, fractional *y*-intercept) over experimental efflux time (minutes) as the forward transfer rate per min (*K*trans, min<sup>1</sup> ) in model selection (Model 1, 2 or 3; [107]). The generalized kinetic model (GKM) is based on Model 3 as the three-parameter model with outward efflux (*K*trans) inoto extracellular tissue space (EES, VD) blood plasma inward rate constant (*K*ep, *K*b) on the Patlak *x*-axis plot; the y- intercept *V*<sup>D</sup> parameter is sometimes required for the model [107]; and there is a strong correlation between the 14C-AIB QAR *K*<sup>i</sup> forward transfer constant (*K*trans, *K*i) and the Gd-DTPA DCE-MRI GKM model *K*trans parameter [108]. DCE-MRI-based bi-compartmental modeling is in *T*1-weighted concentration space (mM) with a linearized signal intensity (SI) to DTPA/DOTA (MW 404 Da)-chelated Gadolinium (Gd, MW 157 Da) concentration determination by the product of the molar relaxivity (*r*<sup>1</sup> 1 ), the longitudinal relaxivity (*R*1), and dynamic imaging SI/SI0 ratio of the low *T*R/*T*<sup>E</sup> dual -flip angle (12O, 3O) FFE sequence-based imaging [64]; and the effective blood-brain/tumor barrier tumor interstitium permeability limit to macromolecular therapeutics is 12 nm as determined by serial DCE-MRI of Gd-DTPA-G1 through -G8 PAMAM dendrimer generations (2–14 nm).

Several studies have compared the GKM kinetic modeling parameters for the detection of the degree of hyperpermeability across tumor types to tumor tissue histopathology, and show high correlation between *K*trans or *V*<sup>e</sup> and lesion grade (*r*, 0.72–0.78) along high probability for dissimilarity with high discriminatory AUC between Grade II and Grade IV lesions (Sens: 100%, Spec: 93.3%) [109]. Other MRIbased parameters include the blood oxygenation level dependent (BOLD) susceptibility weighted imaging (GE SWI) gradient-echo (GE) Δ R2\*plateau tumor signal parameter in the presence of tortuous vascular density (VD) [110], which shows better correlation with microvascular vascular density (MVD, 5–10 μm vessels) and vascular density than the GKM model-derived parameter, and also sensitivity and specificity for distinguishing molecular gene expression marker differences in highgrade glioma (HGG) [111] and could be applicable for other tumor types (i.e. exposure-related). Thus, there is applicability of pharmacokinetic model-based parameter determination for differentiation of earlier grade and higher grade lesions within the range for lower permeability brain neoplasms with DCE imaging (Appendix I), and also could have positive predictive value (PPV) for early detection of non-nervous system peripheral solid tumors in worker populations with comorbid risk factors and higher probability for malignancy.

These further developed models are also applicable for the study of circulatory effects of experimental high-molecular weight, density and size or aspected-size therapeutic agents with an increased probability for risk due to non-selectively and extended half-life-related systemic toxicity. Thus, since bioengineered particulate matter toxicity occurs to tissue capillary walls, and the lining endothelium with secondary alterations to the endothelium epicalyx/glycocalyx layer (EGL) and the subendothelium basement membrane collagen subunits, which are endothelial cellsecreted deposition, these methods can be utilized for initial and serial determinations of toxicity-related alterations at *in vitro*, *in situ* and *in vivo* temporal resolution.

### **6. Geologic detritus particulates**

#### **6.1 Inosilicates**

Asbestos is the double-chain silica tetrahedral-based inosilicate with sharing of two or three atoms each and is comprised of aspected particulate fibers called amphibole fibers with: i) Non-serpentine subtypes Actinolite, Grunerite, Anthophyllite, Crocidolite and Tremolite (<sup>ρ</sup> = 2.58–2.83 gmL<sup>1</sup> ) (**Table 1**), these being the primary and law regulated asbestos fiber subtypes; and ii) the serpentine asbestos form is Chrysotile (*<sup>ρ</sup>* = 2.53 gmL<sup>1</sup> ) within the density interval of the silicates 2.196 gmL<sup>1</sup> (amorphous) - 2.648 gmL<sup>1</sup> (α-quartz). The amphibole asbestos subtypes are defined short fiber (SAF) of less than 5 μm in length, and the long fiber (LAF) asbestos ≥5 μm based on study of Crocidolite, Tremolite, Dawsonite and Wollastonite [31].

Asbestosis disease/mesothelioma, and related lung carcinoma result is an increase in mortality (%) based on an early epidemiologic cohort study of disease-prevalence associated mortality (1950s–70s) in which 17,800 U.S. and Canada trade workers (i.e. welders, shipfitters) were the 10- year prospective cohort for recording of Asbestosis onset radiographic findings in asbestos insulators [32]; i) mesothelioma is associated with a significant increase in mortality over expected in persons with plaques as compared to controls (Ref. [32] Table 8 (*df* = 6), post-hoc X2 *p*-value (*Pr*sig), 0.001; Appendix II. Post-analyses: Chi Square; **Table 6**, under respective table), and there is also a low probability for lung carcinoma-associated increase in mortality that could be significant (Ref. [32] Table 8, X<sup>2</sup> *p*-value, 0.15, α = 0.05); ii) there is an increase in lowest grade abnormalities on X-ray over expected with asbestosis exposure in the 0–9 year and 10–19 year duration of contact Ref. [32] **Table 2** (*df* = 4), post-hoc X<sup>2</sup> *p*values, 0.002, 1.65E-08; **Table 4**), and the 40+ duration of exposure groups postanalysis confirms an decrease in observed Grade I asbestosis X-ray abnormalities with an increase in Grade 2 asbestosis-like abnormalities over expected; **Table 4**, post-hoc X<sup>2</sup> *p*-value, 7.455E-12); and iii) there is shift from fewer observed abnormal X-ray findings to normal in-between 1956 and 1960 and 1961 Ref. [32] Table 10 (*df* = 3), post-hoc X2 *p*-value, 0.0055; **Table 7**). An increase in mortality percent (%; Ref. [32] **Table 5**; data not post-hoc analysed) from lung (bronchogenic) carcinoma is maximal between the 25–29 and 35–39 years of employment exposure strata, and the increase from mesothelioma between the 30–34 and 40–44 years of the same with the skew of the distribution, and the majority in Shipwrights and Boilermakers. The higher mortality from pleural mesothelioma is in the 35–39 year exposure interval, and the higher proportion of peritoneal mesothelioma is over a 45+ period due to metastatic pleural disease. Thus, asbestos exposure results in disease with latency, increased bronchogenic carcinoma earlier and mesothelioma later, in addition to there being the nonlinear increase in risk of lung carcinoma with cigarette smoke co-exposure in workers with asbestosis as reported by the group.

There is an increase in lung tumor and mesothelioma with exposure to >8 μm length particles that have narrower diameters with the highest correlation to tumor for particles with a > 32- to 16- fold aspect ratio, i.e. in length: width strata, > 8 μm ≤ 0.25 μm (*r* = 0.80), and > 4–8 μm ≤ 0.25 μm (*r* = 0.63) [31]; and there is the particle aspect-tumor initiation relationship after deposition by inhalation and trans-

mesothelial peritoneal metastases, or after direct intraperitoneal inoculation in animal models. The other determinant of toxicity appears to be atomic constituency, for example, of some more recently identified asbestos forms (IARC, 2012; [30]), Winchite [(Ca2+, Na1+) Mg2+4 (Al3+, Fe3+, Mn2+) (Si8O22) (OH̄)2] and Richterite [Na1+2, Ca2+) Mg2+ <sup>5</sup> (Si8O22)(OH̄)2], in addition to Crocidolite [Na1+2, Fe2+ 3, Fe3+ 2, Si8O22 (OH̄)2] containing Riebeckite [Na1+2Si8O22(OH̄)2] as the classic common asbestos type as an example of ionic ferrous and/or ferric iron in a polyhedral lattice of silicon oxide. In addition to the presence of covalently bond SiO2 lattice in asbestos fibers, these are comprised of intermolecular ionic bond element interactions (Fe2+, Fe3+), and ferruginous bodies marked by Perl's oxidized iron stain (Fe3+) within tissue interstitium has also been studied in a cumulative exposure cross-sectional study of synchrotron-based x-ray radio-opacity imaging of asbestos bodies with associated Ferritin (variable size), and X-ray fluorescence microscopy (μ-XRF) for elemental composition analysis, which are intracellularly localizing in macrophage-type cells and also present extracellularly (interstitial); and the asbestos body fluorescence analysis reveals high amounts of K > Mg > Na > Fe, and energy absorption analysis (micro-XANES) reveals presence of ferruginous asbestos fiber body constituents, ferrihydrite-containing protein Ferritin (61.7%), and Hematite (22.1%) and Crocidolite (16.2%) [33].

Also, the U.S. Geological Survey asbestos fiber mixtures have now been characterized by aerodynamic equivalent diameter (*D*ae), equivalent diameter (*D*eq) by TEM, and settling velocity (Vt) measurements at elutriator flow funnel settings to capture asbestos fibers ≤2.5 microns, and K-factor atomic number (*Z*)-corrected element composition analysis [36]. Thirteen percent (%) of the mixture is respirable fraction fibers by elutriator recovery at expected respiratory flow (see Appendix I, RPM sampling efficiency); and by STEM mode energy-dispersive x-ray (EDX) spectrum analysis, elemental composition correlations are determined [36]: Fe to Ca or Mg, and Ca to Na ratios are negatively correlated for co-presence, while Mg and Ca, Fe and Na (*r* = 0.573), and K and Na, show positive correlations. The asbestos particle aspect and composition toxicity results in different histopathological subtypes of mesotheliomas includes a breadth of transformed cell types including fusiform (fibrogenic, osteogenic, giant cells) and pleiomorph (medullar, tubulopapillar).

#### **6.2 Silica and inosilicates**

The least soluble particulates include SiO2 and TiO2, and aluminum (III) oxide (Al2O3) is less, and CuO and non-amorphous carbon black (Printex 90) are least, gene expression effects of which have been correlated with solubility; the least soluble result in greater magnitude gene expression effects upon internalization with maintained positive correlation for presence of tissue neutrophils, Saa-1 (liver) and Saa-3 (lung) mRNA [112]. In addition to gene and protein expression responses to bioengineered particle exposure, differences in gene expression between inosilicates, rutile Cristobalite crystalline silica (non-quartz) and amorphous silica concentrations of crystalline silica and Crocidolite asbestos, are studied in comparative cell lines, primary (normal) human bronchial epithelial cell (NHBE) and BEAS 2B on the SV40 virus-integrated epithelial cell line by cDNA hybridization microarray of larger sets of genes and/or qRT-PCR of certain gene sets [34, 38].

The effects of mined Vermiculite deposits (Libby amphibole, LA) composed of a mixture of Winchite (83%), Richterite (11%) and Tremolite (6%) asbestos with TEMbased length: width on primary human airway epithelial cells (HAECs) are studied

with qRT-PCR measurement of inflammatory marker gene expression in normalized dose-response effect (lL-8, IL-6, COX-2, TNF) [35]; and the geometric mean (μ) lengths of the fiber sets are: RTI Amosite, 10.4 μm (*n* = 359), LA2000, 3.34 μm (*n* = 433, width > 1), LA2007 2.47 μm (*n* = 268) and UICC Amosite, 2.07 μm (*n* = 222) [35]. There is a higher potency effect and increase in chemokine IL-8 mRNA (*CXCL8*) levels in response to RTI Amosite particle number dose/cm<sup>2</sup> cell for equivalent concentrations of ionizable iron (inorganic) and chelatable iron (Fe3+) present in both RTI Amosite and UICC Amosite samples as can be determined by ICP-optical emission spectroscopy. The difference in the inflammation-mediated toxicity potential of the fiber sets is due to the prolonged residence time of aspected iron-containing asbestos fiber types inaddition to the probability of the adsorption of oxidized iron forms.

There is methodological validity in same group studies. The findings of two comparative studies with normal human bronchial epithelial cell (NHBE)- and BEAS-2B immortalized normal epithelial cell- types exposed to low- or high-dose crystalline silica, and at 15 and 75 <sup>10</sup><sup>6</sup> <sup>μ</sup>m<sup>2</sup> /cm<sup>2</sup> , or low dose inosilicate (iron-containing asbestos). There are no cell viability differences for low- or high-dose silica-exposed BEAS 2B cells, however there are differences in gene expression by cDNA microarray with shift towards *FOS* (cFOS) and IL-6 related gene expression with Cristobalite silica exposure (p < 0.05/cut off ≥2.0-fold) with reduced false positive risk (FDR 5%) [38], see **Table 1**. The rank order of gene changes between control and respective exposure comparisons is high dose Cristobalite > low dose > Amorphous with little difference in cell adhesion pathway gene activation between the two silica types by gene ontology (GO) molecular pathways correlation analysis; and there are differences in gene expression between exposure between normal and virally-transformed cell types. Low dose Cristobalite silica results in minimal differential gene expression in NHBE cells (3 genes; **Table 1**) [34]; the Asbestos exposure pathway is more towards *JUN*, IL-8 and MMP-1 metalloprotease gene expression by qPCR for specific gene expression, and common silica and asbestos pathway activation includes glucocorticoid (GR), whereas divergent pathways are extracellular matrix synthesis proteoglycan with Crocidolite asbestos exposure, and the Aryl hydrocarbon receptor (*AHR*) pathway with crystalline silica exposure.

#### **6.3 Lead**

Lead is found in steel at 0.15–0.35% concentration in addition to carbon, and is released during the abrasive blasting process, and poses a risk, as there is little positive or negative correlation and independence to air flow velocity and there is an increase in exposure-related Lead concentration in blood in both blaster and vacuumer [113]. There is the effect of exposure during the ore lead extraction smelting process and to combusted lead particulates over prolonged duration during which dissolution favors ionic Lead (Pb2+) release. There is an increase in the stratified years interval SMR (1– 5 yr., 5–20 yr., 20+ yr) for renal carcinoma and non-malignant respiratory disease (emphysema, pneumoconiosis) [46]: this is in employees with BLL at 56.3 μg/dL (2.73 μM) and 0.366 ppm average airborne Lead concentration exposure (3.1 mg/m<sup>3</sup> ; **Table 3**, Appendix III. Particulates toxicity industrial hygiene), but is with low probability for anemia (10%), in a low co-exposure to Arsenic (0.0133 ppm) and Cadmium (0.0016 ppm) population of workers with unknown smoking history. Inaddition to accumulation levels in long bone parts (i.e. tibia, 99.4 μM), there is a negative *x-* (patella Pb, μg/g), *y*- (LINE-1 methylation) variable regression

relationship for increasing Lead levels to 40 μg/g bone concentration (0.194 mM) that is consistent with decreased inactive LINE-1 DNA and RT DNA methylation with increased Pb concentration; this evidence comes from a cross-sectional study (2010) of global DNA CpG island methylation of retrotransposon viral origin human DNA integrated long repetitive base sequence elements (LINE-1) and reverse transcriptase (RT)-encoding elements [114].

There are also increases in inflammatory marker expression (IL-6) inaddition to of both vascular permeability factor gene (*VEGFA*) and decoy receptor (sVegfr1) gene expression as per another more recent single timepoint study (2017) enrolled Lead-Zinc workers with elevated BLL (37 μg/dL) and ZPP levels as compared to controls with unknown alcohol use history [47]. Additional support for the Lead transition metaltype toxicity effect comes from cell culture exposure with applied Lead Acetate (100 nM – 1 μM; **Table 1**) on transiently-transfected chondrocytes with measured reporter luciferase (LUC) activity of AP-1 and Nf-KB plasmids in response to Pb (C2H3O2)2 alone or with applied PTHrP [50], in which its application alone results in a decrease in cell survival factor gene Nf-KB without change in AP-1 factor subunit genes (*FOS*, *JUN*) activity, but an increase in AP-1 activity with PTHrP as effect modifier. The effect of released ionic Pb is condition-dependent and can in certain instances shift the cell response towards pro-angiogenic progression. The effect of released ionic Lead (Pb2+) is co-exposure condition-dependent, and can in certain cell types and instances shift the cell response towards pro-angiogenic phenotype, inaddition to its known negative effects on neuronal cell growth in association with Casp8 gene promoter hypomethylation (CpG) [48] and over activation resultant neurotoxicity.

#### **6.4 Manganese**

Manganese has paramagnetic properties, and is present in various oxidation states, Mn2+ (Mn(OH)2, basic), Mn3+ (Mn2O3, Mn(OH)3) or Mn4+ (MnO2) in the presence of H2O2 reactive Oxygen species (ROS) with co-product hydroxides formed (OH, OH) as per the Pourbaix electrical potential to pH relationship [115]. It is reactive with Transferrin as Mn3+ in the presence of base (HCO3 ) similar to that of ferric iron, but with less affinity than ionic Chromium (Cr3+) due to a greater ratio of bicarbonate to metal as determined by optical absorbance [116], and as shown by the Cr (OH)x-Transferrin peak on EPR spectroscopy [116]. An example of exposure to particulate matter metal mixture is in welding with fume generation, and dissimilar solid or flux-cored pre-weld composition-containing wire-dependent secondary aerosolizing particulates of differing solubilities and toxicity.

These hazardous by-products of welding have been characterized [117]: i) the size distribution of particles generated is between 3 to 180 nm (SEM, TEM); ii) the mixture in a) solid wire gas metal arc welding (GMAW; S1, S2) is a combination of oxides, FexOy, MnxOy, CrO4 <sup>2</sup> (CrVI), Cr2O3 (CrIII/IV), SiO2 and BiO2, and in b) fluxcored wire arc welding (FCAW; F1, F2), this includes the additional composition of ionically-bonded Alkalis (Na<sup>+</sup> , K<sup>+</sup> ; Group 1, Period 3, 4) and Halogen (F; Group 17, Period 2) elements (XPS); and iii) the PBS soluble metal is a small proportion in solid wire welding, while in flux wire welding, it is most of the (by-) product comprised of Mn, CrIII and CrVI, predominantly CrO4 <sup>2</sup> (CrVI). The weld by the FCAW process has flux by-product with increased solubility and the weld proportion of metal product content improved in purity with less metal oxide, but there is a large proportion percentage (%) with higher *Pr* for sig. For concentration-dependent cytotoxicity at 1 day and genotoxicity as early as 3 hrs at 10<sup>4</sup> M concentration in the flux FCAW F1

and F2 process type welding [117]. Thus, any combustion by-product composition has Manganese in its ionic (Mn2+) form, which is the energetically-favorable form at pH < 8, in both sub-type processes (S2, F2), which is supported by the experimental data showing solubility decreases with addition of increasing concentration of Fluorine (F).

As to the Manganese effects on genomics, DNA re-sequencing results shows Manganese efflux transporter gene *SLC30A10* and *SlC39A14* polymorphism ([118]; **Table 1**), and changes result in other related gene expression such as *RBFOX1* [119], *TXN8* [48] and MPP+ responsive influx transporter gene *DMT1* [120], for example bisulfite Cytosine ( > Uridine) substitution DNA epigenomics shows methylation of the splicing regulator gene *RBFOX1* (A2BP1) at position cg02042823, which has been studied by qT-PCR and RNA-seq transcriptomics. The double-allele mutant *lox*ed gene (*RBFOX1*/) murines show a decrease in exon inclusion for certain channel genes such *GABRG2* and *GRIN1* [119] with the net effect being lower stimulus intensity thresholds required to evoke EPSPs and less inhibitory ion flux as per potential recordings showing this effect.

In addition to study of alterations in gene methylation of the *RBFOX1* gene, study of cell morphology shows altered gender-dimorphic dendritic spine count with spatial frequency lower in females (10 μm<sup>1</sup> ), and higher in males, after subcutaneous exposure to repeated dosing of ionic Manganese as MnCl2 [121] such that this would result in Dopaminergic with a sex-specific dimorphic effect on neurite bleb budding in association with dendrite density. Similar local concentrations achieved in the striatum in both and the inverse inhalation effect of Maghemite (Fe2O3) in the Welder workers [122] with the clinical sign of cocked-gait as sign of basal ganglia and midbrain nuclear group toxicity with internal capsule upper motor neuron disinhibition effects due to exposure intensity toxicity early in Manganism, and then exposure duration-related toxicity in Manganese-induced Parkinsonism).

The experimental data on the transfected overexpression of the divalent influx transporter gene, *DMT1*, that transports Mn2+ present at increased local concentration in SH-SY5Y pre-differentiated neuroblastoma neuronal cell line show the effects of Manganese (Mn2+) in comparison to ferrous Iron (Fe2+) with more of an initial shift in cell compliance (*P*eff C, a.u.) to the phosphorylation of the c-JUN AP1 transcription factor terminal kinases [123], and towards the resultant p53-mediated R-shift for activation of the *Bax-*, *Bad-* and *Bim-* apoptosis cascade, and/or DNA repair and cell cycle changes. For Manganese, i) the negative electrical potential and neutral pH favors its ionic and soluble form; and ii) it is the presence of efflux channel and certain cell surface internalization receptors that results in its initial effect followed by its later apoptosis-related toxicity to basal ganglia cell types.

#### **6.5 Copper ore arsenate**

Either Arsenic or Stibium, Group 15 (semimetals), are bound to Sulfur (Group 16, nonmetal), and there is 91% dissolution of Tennantite at high temperature (212° F) over a 2 hour period in basic electrolyte solution (NaOH) containing Na2S [124]. The mined copper ore contains transition metals, Copper, Arsenic and Iron in polyhedral/ hextetrahedral configuration with some forms containing ionic free forms of Iron (Fe3+/2+) and Zinc (Zn2+) (**Table 1**). Copper in oxidation states 1<sup>+</sup> or 2+ results in interactions with metallothioneins with overload effect on DNA, resulting in conformational changes [125]. Certain studies show the effect of ionic Copper in form of Cu (II) acetate ((CH3)2COO-)2 on cells that when applied results in overexpression of the

NFκB as per plasmid transfectant fluorescence and of pathway and related genes as determined by qRT-PCR and confirmed by siRNA data [43]. Copper (Cupric) exposure to cells (i.e. Hepatoma G2) results in a time-dependent increase in NFκB2 target gene *CXCL8* (IL-8) and *IER3* gene expression, inaddition to overexpression of *NFE2L2* (NRF-2) and Phase II detoxification pathway genes, *HMOX1* (HO-1) and *GCLC*. In desaturation conditions (Section IV), the dissolution kinetics favor conversion of As2S3 to H3ASO3 and hydrogen sulfide gas (H2S); Arsenate (AsO4) <sup>3</sup> and Arsenite (AsO3) <sup>3</sup> in respective trivalent and pentavalent oxidation states are slow dissolution molecules with the potential to transform cells in low-chronic exposure conditions with ROS pathway and related gene activation [126], and hypoxiaassociated genes (i.e. HIF-1α) in transformed cells [44]. In the Chile Copper ore subregions (various forms, see **Table 1**) with mining area-specific differences in exposure risk [127], there is increased gender-specific mortality in a low-smoke exposure prevalence population of men and women ≥30 years of age as analysis by the age-weighted standard mortality ratio (SMR; [14]) [45] supports increased mortality due to carcinomas of the bladder (SMR 6.0, M; 8.2, F), kidney (SMR, 1.6, M; 2.7, F), pulmonary (SMR, 3.8, M; 3.1) and dermatologic (SMR, 7.7, M; 3.2, F), and the population attributable risk (PAR) in that mining region is 9.7% (M) and 4.9% (F) for ore exposurerelated carcinoma. Leaching into drinking water reservoirs with peak exposure concentration at 870 ug/dL (116 μM) results in an increase in mortality due to lung cancer is attributable to increased ore particulates air concentration. The presence of additional transitional metals, Iron, Stibium and Zinc present in Copper ore particulates could result in effect modification.

### **7. Exposure prevention and surveillance guidelines**

In the various sectors there are increased levels of process-generated smoke particulates or aerosol mists and related hazards that require the implementation of the hierarchy of controls and access to online information repositories [128]. Surveillance of occupationally-associated promontory habits prevalence has shown co-existence of comorbidity such as in the asbestos abatement workers [129]. Chest X-ray radioopacities are present in individuals not exposed to dusts in an intercontinental population with gender- and age- specific differences with low prevalence at ≤50 yoa, in females (F, 0.4%) or blue collar workers working in non-exposure conditions (0.21– 0.25%) [130]. Exposure surveillance comprises spirometry and CXR for monitoring of both PMF and simple CWP (under MSHA (30 USC 801–962) with a PEL of 2 mg/m3 (REL 1 mg/m<sup>3</sup> ) or reduced PEL for silica content (30 CFR 70.101, see **Table 4**, Appendix III), and the requirement of a Black Lung (B) read (42 CFR Part 37) as per ILO classification for radiographs (1980) [131], **Table 1**. In the particulates exposuremonitored individuals with FEV1/FVC ratio increases due to silica or asbestos exposure, there is a higher probability for parenchymal abnormality by age and Laborer/ Cleaners class (Model I), but not in an un-conditional Model (II) without inclusion of asbestos exposure in logit probability [132].

Surveillance measures can be proposed for higher risk work groups in similar exposure conditions, and at the action limit there is the requirement for substancespecific NIOSH-approved respiratory protective device use under the OSHA Respiratory Protection Standard (29 CFR 1910.134). There is environmental risk for carcinogenesis with air exposure concentration at 8.40x10<sup>2</sup> (CrVI) and majority of years of life lost due to water exposure to Free Cr6+ (2.09x10<sup>5</sup> ; YLLwater, 92%) [133] [REF]

with the effective cancer risk at two-orders higher concentration for the particulate, aerosol and water partitioned agents tested (i.e. HgII, CdII; Lindane, DDT). There is increased risk for lung carcinoma in certain worker sub-groups such as Crane, Derrick and Hoist workers (ORadj 14.4; 3.36% PAR), and with ≥10 years exposure to Coal Dust (IARC 1) adjusted for other variables shows greater odds of lung cancer in cases over non-cancer (ORadj 2.0) and cancer (ORadj 1.5) control group populations [134], as per a 1992 two-control group case-control study applying multi-coefficient variable probability regression (unconditional) modeling; as per the study, any duration of cigarette smoke exposure ≥20/day results in increased risk of lung carcinoma (ORadj 2.1) in Asbestos–exposed persons.

Area sampling of air for aerosolized viruses transmitted by speak, cough or sneeze [54] and particulates is by general elutriation devices [16], carbon-filled adsorption capillary tubes for lipophilic gaseous vapors, or personal dosimeter devices with pore size threshold filters (**Table 5**, Appendix III), to adhere to certain exposure threshold monitoring requirements at the action level (AL) to begin biomonitoring. The OSHA air concentration medical action level for Lead (PbII, Pb2+, IARC 2A; MW = 206.2 Da) is 0.03 mg/m3 (10<sup>10</sup> M) with a PEL (ceiling limit) of 50 ug/m<sup>3</sup> under 29 CFR 1910.1025 (https://www.osha.gov/laws-regs), and Manganese (MnII) has a threshold limit value (TLV, respiratory limit)-8 hr. TWA of 0.02 mg/m3 as recommended by the American Conference of Governmental Industrial Hygienists (ACGIH) [15]. Occupational surveillance of inhalable dust exposure includes for IARC Group I carcinogens, Asbestos (29 CFR 1910.1001, Industry), Silica (29 CFR 1926.1153, Construction), Chromium (CrO3, CrVI; 1910.1026, GI), and also Arsenic (ArV, inorganic; 29 CFR 1910.1018) for which there exists a permissible exposure limit (PEL) of 10 ug/m3 with an AL that is one-half of this limit.

Traditional X-ray diffraction has sufficient sensitivity for detection of high-Hounsfield unit pathology in linear scale with exposure monitoring reads as per the ILO reads classification system [131], and has high specificity to detect early metastatic foci (91%), but low sensitivity (41%) [135]. Non-contrast chest CT can be utilized for study temporal tracking of pulmonary lesions at high-resolution as it has improved sensitivity (74%). *T*1-weighted MRI at early time point after injection early contrast enhancement (ECE; 4.5 min) can be utilized for signal intensity (SI)-based hyper-permeability detection of mesothelioma foci by region of interest (ROI) as it has high sensitivity (91%) and 15% false positive rate with a high-correlation κ statistic (0.77) [136]; and 18FDG PET has utility for quantitative detection of solid tumor tissue hypermetabolism and metastatic disease with a high true positive rate (92%) and high specificity (93%) [135]. The diagnostic determination of occupationally-related neoplasia can include the application of macromolecular contrast enhancement agents for sensitive and specific detection of the hyperpermeable pathology of solid tumors on CT and on MRI during surveillance or treatment course.
