**4. Summary and conclusion**

380 Toxicity and Drug Testing

expression of human SULT1A1, but not in control cells. 2-Amino-3-methylimidazo[4,5-f] quinoline induced gene mutations in V79 cells co-expressing human NAT2 or NAT1 together with human CYP1A2, even at a concentration of 0.01 and 1 μM, respectively, but was inactive (even at 30 μM) in cells expressing only CYP1A2 (Glatt, 2005; Glatt, 2006). Genotoxicity is a branch of the field of toxicology that assesses the effects of chemicals on DNA or genetic processes of living cells. Such effects can be accessed directly by measuring the interaction of chemicals with DNA or more indirectly through the production of gene mutation or chromosome alterations. The observations of these consequences in the genotoxicity tests suggest the carcinogenic concern of a chemical. Thus it is important to improve the genotoxicity test system to evaluate accurately based on the in vivo situation in human as much as possible. In the present research, I tried to imitate human metabolism by using human hepatocyte cell line expressing human CYP enzymes. My results indicated that metabolism focused only on CYP was not sufficient to evaluate the genotoxicity of the chemicals such as tamoxifen and safrole. A comprehensive metabolic pathway not only by phase I drug-metabolizing enzymes but also by phase II enzymes would be needed for the accurate assessment of genotoxicity. Moreover, other cellular defense systems (i.e., antioxidant system, GSH or ascorbic acid, and DNA repair system) are involved in the expression of genotoxicity by a chemical. Despite the proof that most chemical carcinogens undergo metabolic conversion into DNA-reactive intermediates, some compounds do not bind to DNA and are not mutagenic, yet they are carcinogenic in animal models and possibly also in humans. These non-genotoxic mechanisms such as induction of inflammation, immunosuppression, formation of reactive oxygen species, activation of receptors such as arylhydrocarbon receptor or estrogen receptor, and epigenetic silencing. Therefore ,another approach based on the non-genotoxic mechanism is necessary to predict the carcinogenic action from a certain chemical. Together, these genotoxic and nongenotoxic mechanisms can alter signal-transduction pathways that finally result in hypermutability, genomic instability, loss of proliferation control, and resistance to apoptosis — some of the characteristic features of cancer cells. In this regard, we need to learn much more about the role and interplay of susceptibility and resistance function targeted by human carcinogens or involved in modulating human responses to carcinogenic

Genotoxicity is a branch of the field of toxicology that assesses the effects of chemicals on DNA or genetic processes of living cells. Such effects can be accessed directly by measuring the interaction of chemicals with DNA or more indirectly through the production of gene mutation or chromosome alterations. The observations of these consequences in the genotoxicity tests suggest the carcinogenic concern of a chemical. Thus it is important to improve the genotoxicity test system to evaluate accurately based on the in vivo situation in human as much as possible. In the present research, I tried to imitate human metabolism by using human hepatocyte cell line expressing human CYP enzymes. My results indicated that metabolism focused only on CYP was not sufficient to evaluate the genotoxicity of the chemicals such as tamoxifen and safrole. A comprehensive metabolic pathway not only by phase I drug-metabolizing enzymes but also by phase II enzymes would be needed for the accurate assessment of genotoxicity. Moreover, other cellular defense systems (i.e., antioxidant system, GSH or ascorbic acid, and DNA repair system) are involved in the

chemicals.

**3. Future considerations** 

Many carcinogens are known to be procarcinogens and require metabolic activation to exert their genotoxicity through the formation of reactive intermediates. Therefore, for hazard identification on the genotoxic potential of drug candidate and its metabolites, S9 fraction prepared from the livers of rats pretreated with phenobarbital and 5,6-benzoflavone or with Aroclor 1254 to induce drug-metabolizing enzyme activity must be used in the in vitro genotoxicity testing. However, it is frequently questioned as to whether such an in vitro metabolite generation system is the most relevant for human risk, or whether the assay would be better served by using a human-derived in vitro system. In the present study, we examined the advantages of HepG2 transformants expressing a series of human CYP isoforms as a better alternative for metabolic activation system in the in vitro genotoxicity testing.

In section 2.1, the sensitivity of this system to detect genotoxicity requiring CYP activation was confirmed in the in vitro micronucleus tests using well-studied model chemicals. These results showed HepG2 transformants system have the appropriate sensitivity to detect genotoxicity requiring CYP activation tests using well-studied model chemicals. In addition, based on results obtained in the DMBA and ifosfamide treatments, HepG2 transformant system showed that genotoxic metabolites would be produced by not only the most active CYP isoform but also by other less active CYPs.

In chapter 2.2, this system allowed us to investigate the genotoxicity of model chemicals for which the contributing CYP isoforms, especially those mediated by CYP1A2 or 3A4 which is known to metabolize many drugs in humans, have not yet been identified. Based on the results obtained in the okadaic acid and β-endosulfan treatments, it was clearly demonstrated that the HepG2 transformant system was able to identify the CYP isoform relating to the genotoxicity of chemical metabolite(s) and was useful to elucidate the genotoxicity of a new chemical or a drug candidate in the presence of the metabolic activation system.

In chapter 2.3, the relevance of the interaction between phase I and phase II drugmetabolizing enzymes, e.g., UGT, GST, and SULT, in the test system was demonstrated in a MN test of tamoxifen or safrole, which has been reported to be metabolized by enzymes of both phases. Based on the results for tamoxifen and safrole, it was clearly demonstrated that the interaction between the phase I and phase II drug-metabolizing enzymes was crucial to

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**17** 

*Iran* 

**Genotoxic Impurities in Pharmaceuticals** 

Genotoxic compounds induce genetic mutations and/or chromosomal rearrangements and can therefore act as carcinogenic compounds (McGovern and Jacobson-Kram, 2006). These compounds cause damage to DNA by different mechanisms such as alkylation or other interactions that can lead to mutation of the genetic codes. In general, chemists employ the terms "genotoxic" and "mutagenic" synonymously; however, there is a subtle distinction. Genotoxicity pertains to all types of DNA damage (including mutagenicity), whereas mutagenicity pertains specifically to mutation induction at the gene and chromosome levels. Thus, the term "genotoxic" is applied to agents that interact with DNA and/or its associated cellular components (e.g. the spindle apparatus) or enzymes (e.g. topoisomerases) (Dearfield *et al.*, 2002; Robinson, 2010). Irrespective of the mechanism by which cancer is induced, it is now well agreed that it involves a change in the integrity or expression of genomic DNA. The majority of chemical carcinogens are capable of causing DNA damage, i.e., are "genotoxic" (Ashby, 1990). Moreover, a genotoxic compound also carries with it the

carcinogenic effect which causes additional concern from the safety viewpoint.

Drug substances and their relative compounds such as impurities constitute an important group of genotoxic compounds. Thus, these compounds pose an additive concern to clinical subjects and patients (Müller *et al.*, 2006). Considering the importance of this problem, the challenge for regulatory agencies is to form guidelines and standards for the identification and control of genotoxic compounds and their impurities especially in pharmaceuticals. In this article, genotoxicity profiles of the main group of genotoxic compounds are discussed. The article throws light on the challenges in analyzing and predicting for these groups and also deals with the different management problems of genotoxic impurities in

The International Conference on Harmonization (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use project represents the main group of guidelines with topics such as "Quality" topics and "Safety" topics. Quality topics relate to chemical and pharmaceutical quality assurance (stability testing, impurity testing, etc.) and

**1. Introduction** 

pharmaceuticals.

**2. Guidelines 2.1 ICH guidelines**  Abolghasem Jouyban1 and Hamed Parsa2

*2Tuberculosis and Lung Disease Research Center, Tabriz University of Medical Sciences, Tabriz,* 

*1Drug Applied Research Center and Faculty of Pharmacy,* 

