**2. Effects of spaceflight on salivary glands**

#### **2.1 Morphology**

The parenchyma of salivary glands consists of a collection of secretory endpieces, or acini, connected to a ductal system that conveys saliva to the oral cavity [10, 11] (**Figure 1A**). The acini produce the primary saliva containing the majority of the proteins, most of the electrolytes and all of the water present in the final saliva. Serous acinar cells (**Figure 1B**) produce proteins and glycoproteins; seen in transmission electron micrographs (TEMs) the content of their secretory granules has a moderate to high density. Mucous acinar cells produce mainly mucins; their secretory granules exhibit a low density and often are fused with adjacent granules. The acinar secretory cells form a roughly spherical structure, their bases resting on the interstitial connective tissue, their apices facing a central lumen, and adjacent cells are joined by junctional complexes separating the interstitial from the luminal compartments. The cells contain abundant rough endoplasmic reticulum (RER), a prominent Golgi complex, and numerous secretory granules in the supranuclear cytoplasm. Secretory proteins synthesized by the cells are stored in the granules until their contents are released into the lumen by exocytosis. Consequently, analysis of the glandular tissue provides information about the composition of saliva.

Saliva produced in the acini flows into the initial portion of the ductal system, the intercalated ducts. These small ducts merge, forming larger ducts that

**55**

tion of Na<sup>+</sup>

**Figure 1.**

that empties into the oral cavity.

*from ([10], Chapter 11, pp. 224, 225)).*

*Oral Tissue Responses to Travel in Space DOI: http://dx.doi.org/10.5772/intechopen.86728*

eventually empty into the main ductal component, the striated ducts. The cells of these ducts are columnar in shape, and have abundant mitochondria nestled between infoldings of the basal cell membrane. Primary saliva produced by the acinar cells is modified by the striated (and excretory) ducts principally by reabsorp-

*(A) Diagram showing the main parenchymal components of mammalian salivary glands. (B) Transmission electron micrograph (TEM) of a serous acinar cell of a rodent parotid gland (PG) (modified, with permission,* 

a pH near neutrality. The striated ducts become excretory ducts as they enter the connective tissue septa that partition the gland into lobules and lobes. The excretory ducts gradually merge into larger ducts and finally form the main excretory duct

In humans, the acinar cells of the parotid gland (PG) are all serous. The submandibular gland (SMG) is a mixed gland, with mostly serous acini but also some mucous acini that are capped by serous cells forming a structure called a demilune. The sublingual gland (SLG) also is a mixed gland; its acini are predominantly mucous with serous demilunes. The acini and ductal systems of the rodent PG and SLG are very similar to those of the human (**Figure 2**). The structure of the rodent SMG (**Figure 3**) differs significantly from that of the human. The acini in rodents are all seromucous and produce a number of proteins and glycoproteins as well as mucins. As the animals become sexually mature, the cells of first portion of the striated ducts

and Cl<sup>−</sup> and addition of a few proteins; the final saliva is hypotonic with

*Oral Tissue Responses to Travel in Space DOI: http://dx.doi.org/10.5772/intechopen.86728*

*Beyond LEO - Human Health Issues for Deep Space Exploration*

assess physiological and environmental stressors [2].

of the physiological status of astronauts.

**2. Effects of spaceflight on salivary glands**

**2.1 Morphology**

Among the organic constituents of saliva are digestive enzymes, calcium binding proteins, a variety of growth factors and regulatory molecules, antimicrobial components and immunoglobulins, and mucins that lubricate and moisten the oral tissues [1]. Other protective components of saliva include those involved in buffering and neutralizing acids produced by oral microorganisms and ingested with food and drink. Most are produced and secreted by the cells of the salivary glands, nevertheless a number of other proteins derived from other cells, tissues and organs also find their way into saliva. The presence of these substances and the ease of, and the noninvasive means of, collecting saliva have led to a great deal of interest for its use as a diagnostic fluid. Consequently, significant progress has been made in using saliva to detect oral and other cancers, several oral and systemic diseases, and to

Our early studies of rats flown on Spacelab-3 [3, 4] indicated that exposure to microgravity resulted in specific changes in salivary gland structure and biochemistry. Accordingly, the premise for our recent studies of mice flown on the US space shuttles and the Russian Bion-M1 biosatellite, was that changes in the expression of salivary gland secretory proteins appear to occur in microgravity, and that the pattern of changes detectable in saliva could be employed to assess important aspects

In addition to studying salivary gland tissues, we examined the effects of microgravity on the mandibles and teeth of mice flown in space. Numerous studies have been carried out documenting the effects of spaceflight on bones of the weightbearing skeleton, especially the bones of the lower limbs and vertebrae (see reviews in [5–7]). However, only a few studies have focused on non-weight-bearing bones, such as the mandible and cranial bones, and teeth [8, 9]. Teeth, once formed, are relatively stable structures, but can be affected by changes in the oral environment. An understanding of the effects of microgravity on tooth development, which can be studied using the continuously erupting rodent incisor, is important especially in view of future possibilities of long-term space journeys and colonization of other planets.

The parenchyma of salivary glands consists of a collection of secretory endpieces, or acini, connected to a ductal system that conveys saliva to the oral cavity [10, 11] (**Figure 1A**). The acini produce the primary saliva containing the majority of the proteins, most of the electrolytes and all of the water present in the final saliva. Serous acinar cells (**Figure 1B**) produce proteins and glycoproteins; seen in transmission electron micrographs (TEMs) the content of their secretory granules has a moderate to high density. Mucous acinar cells produce mainly mucins; their secretory granules exhibit a low density and often are fused with adjacent granules. The acinar secretory cells form a roughly spherical structure, their bases resting on the interstitial connective tissue, their apices facing a central lumen, and adjacent cells are joined by junctional complexes separating the interstitial from the luminal compartments. The cells contain abundant rough endoplasmic reticulum (RER), a prominent Golgi complex, and numerous secretory granules in the supranuclear cytoplasm. Secretory proteins synthesized by the cells are stored in the granules until their contents are released into the lumen by exocytosis. Consequently, analysis of the glandular tissue provides information about the composition of saliva. Saliva produced in the acini flows into the initial portion of the ductal system, the intercalated ducts. These small ducts merge, forming larger ducts that

**54**

#### **Figure 1.**

*(A) Diagram showing the main parenchymal components of mammalian salivary glands. (B) Transmission electron micrograph (TEM) of a serous acinar cell of a rodent parotid gland (PG) (modified, with permission, from ([10], Chapter 11, pp. 224, 225)).*

eventually empty into the main ductal component, the striated ducts. The cells of these ducts are columnar in shape, and have abundant mitochondria nestled between infoldings of the basal cell membrane. Primary saliva produced by the acinar cells is modified by the striated (and excretory) ducts principally by reabsorption of Na<sup>+</sup> and Cl<sup>−</sup> and addition of a few proteins; the final saliva is hypotonic with a pH near neutrality. The striated ducts become excretory ducts as they enter the connective tissue septa that partition the gland into lobules and lobes. The excretory ducts gradually merge into larger ducts and finally form the main excretory duct that empties into the oral cavity.

In humans, the acinar cells of the parotid gland (PG) are all serous. The submandibular gland (SMG) is a mixed gland, with mostly serous acini but also some mucous acini that are capped by serous cells forming a structure called a demilune. The sublingual gland (SLG) also is a mixed gland; its acini are predominantly mucous with serous demilunes. The acini and ductal systems of the rodent PG and SLG are very similar to those of the human (**Figure 2**). The structure of the rodent SMG (**Figure 3**) differs significantly from that of the human. The acini in rodents are all seromucous and produce a number of proteins and glycoproteins as well as mucins. As the animals become sexually mature, the cells of first portion of the striated ducts

#### **Figure 2.**

*TEMs of (A) PG serous acinar cells from a STS-135 flight mouse, and (B) sublingual gland (SLG) mucous acinar cells and serous demilune cells from a STS-131 habitat ground control mouse. Mucous acinar cells are filled with electron lucent mucous granules (MG). Serous demilune cells (lower right) contain electron dense secretory granules (SG). Lumen (L), mitochondria (M), nucleus (N), rough endoplasmic reticulum (RER), Golgi complex (arrowheads), intercellular canaliculi (arrows).*

enlarge and synthesize a number of growth factors and proteases that are stored in large secretory granules in the apical cytoplasm. The development of this portion of the duct, called the granular convoluted duct, is strongly influenced by androgens; the granular convoluted duct is, therefore, more prominent in males than in females. Another feature of the sexual dimorphism seen in the rodent SMG is the presence in females of terminal tubule cells, or granular intercalated duct cells, at the acinarintercalated duct junction. These cells are remnants of the early development of the gland; in males they are entirely eliminated by apoptosis by about 1 month of age.

Specific morphological changes were seen in the PG after a 13- to 15-day flight on the STS-135 and STS-131 shuttle missions, and 30 days on the Bion-M1 biosatellite [12, 13]. In the acinar cells autophagic vacuoles were common, and apoptotic

**57**

**Figure 3.**

cells were seen more frequently (**Figure 4A**). The autophagic vacuoles often contained degenerating secretory granules as well as other organelles. Some cells in the intercalated and striated ducts had large endocytic vacuoles containing dense content in the apical cytoplasm (**Figure 4B**). Immunogold labeling showed the

*TEMs of submandibular gland (SMG). (A) seromucous acinar cell from a STS-131 flight mouse, (B) intercalated duct and terminal tubule cell from a STS-131 habitat control mouse, and (C) granular convoluted duct from a Bion-M1 flight mouse. Acinar cells have a basal nucleus (N) and RER, and large secretory granules (SG) with a granular content of low density. In female mice, terminal tubule cells (TT) often are present in the intercalated ducts (ID) adjacent to the acinar cells. Columnar shaped granular convoluted duct cells of the male gland have a basal nucleus and mitochondria, and numerous dense secretory granules in the supranuclear cytoplasm. Lumen (L), mitochondria (M), Golgi complex (arrowheads), intercellular canaliculus (arrow).*

presence of acinar secretory proteins in these vacuoles.

*Oral Tissue Responses to Travel in Space DOI: http://dx.doi.org/10.5772/intechopen.86728* *Beyond LEO - Human Health Issues for Deep Space Exploration*

enlarge and synthesize a number of growth factors and proteases that are stored in large secretory granules in the apical cytoplasm. The development of this portion of the duct, called the granular convoluted duct, is strongly influenced by androgens; the granular convoluted duct is, therefore, more prominent in males than in females. Another feature of the sexual dimorphism seen in the rodent SMG is the presence in females of terminal tubule cells, or granular intercalated duct cells, at the acinarintercalated duct junction. These cells are remnants of the early development of the gland; in males they are entirely eliminated by apoptosis by about 1 month of age. Specific morphological changes were seen in the PG after a 13- to 15-day flight on the STS-135 and STS-131 shuttle missions, and 30 days on the Bion-M1 biosatellite [12, 13]. In the acinar cells autophagic vacuoles were common, and apoptotic

*Golgi complex (arrowheads), intercellular canaliculi (arrows).*

*TEMs of (A) PG serous acinar cells from a STS-135 flight mouse, and (B) sublingual gland (SLG) mucous acinar cells and serous demilune cells from a STS-131 habitat ground control mouse. Mucous acinar cells are filled with electron lucent mucous granules (MG). Serous demilune cells (lower right) contain electron dense secretory granules (SG). Lumen (L), mitochondria (M), nucleus (N), rough endoplasmic reticulum (RER),* 

**56**

**Figure 2.**

#### **Figure 3.**

*TEMs of submandibular gland (SMG). (A) seromucous acinar cell from a STS-131 flight mouse, (B) intercalated duct and terminal tubule cell from a STS-131 habitat control mouse, and (C) granular convoluted duct from a Bion-M1 flight mouse. Acinar cells have a basal nucleus (N) and RER, and large secretory granules (SG) with a granular content of low density. In female mice, terminal tubule cells (TT) often are present in the intercalated ducts (ID) adjacent to the acinar cells. Columnar shaped granular convoluted duct cells of the male gland have a basal nucleus and mitochondria, and numerous dense secretory granules in the supranuclear cytoplasm. Lumen (L), mitochondria (M), Golgi complex (arrowheads), intercellular canaliculus (arrow).*

cells were seen more frequently (**Figure 4A**). The autophagic vacuoles often contained degenerating secretory granules as well as other organelles. Some cells in the intercalated and striated ducts had large endocytic vacuoles containing dense content in the apical cytoplasm (**Figure 4B**). Immunogold labeling showed the presence of acinar secretory proteins in these vacuoles.

#### **Figure 4.**

*TEMs of PG from STS-135 flight mice. (A) An acinar cell contains 2 autophagic vacuoles (AV), and a macrophage (Mac) has engulfed an apoptotic acinar cell (APOP). (B) Intercalated duct cells contain apical vacuoles (arrows) resulting from endocytosis of secretory proteins from the lumen (L) (with permission, from [13]).*

The underlying basis for these changes is believed at least in part to be the loss of neural stimulation due to reduced masticatory activity. This is indicated by experiments where withholding food for 24–48 h, or feeding rats a liquid diet, results in numerous autophagic vacuoles containing degenerating secretory granules and an increase in the number of apoptotic cells in the PG [14–17].

The mice from the Bion-M1 mission all gained weight during the flight but were fed a soft paste diet. The mice from the space shuttle missions had *ad libitum* access to NASA rodent food bars [18], but lost a small amount of weight, as did the habitat control mice (maintained on the ground for the same length of time in the same habitats as the flight mice). This suggests that the mice on the space shuttle flights may not have eaten the amount of food sufficient to maintain their body weight. Since the number of autophagic vacuoles and frequency of apoptosis was greater in flight mice than in the habitat controls, microgravity also appears to affect these processes.

Endocytosis of salivary proteins by duct cells occurs in experimental diabetes [19, 20]. This suggests that the proteins may have an altered structure that is recognized as foreign by the duct cells; these cells are capable of endocytosing foreign proteins introduced in a retrograde fashion *via* the main excretory duct [21–23]. Alternatively, duct cell function in the flight mice may have been altered.

In contrast to the PG, no morphological changes were evident in the SMG or SLG of the flight mice. Similarly, feeding a liquid diet has no apparent effect on the morphology of the rat SMG and SLG [24].

#### **2.2 Salivary protein expression studied using two major approaches**

#### *2.2.1 Electron microscopic immunogold labeling*

Relative changes in secretory protein expression can be analyzed by electron microscopic immunogold labeling. Determination of the labeling density (gold particles/μm2 )

**59**

**Figure 5.**

*+, p = 0.065; \*, p < 0.05; #, p < 0.01.*

*Oral Tissue Responses to Travel in Space DOI: http://dx.doi.org/10.5772/intechopen.86728*

of secretory granules of PG acinar and duct cells showed alterations in the expression of

*TEM immunogold labeling for proline-rich protein (PRP) in PG acinar cells of (A) flight and (B) habitat ground control mice from STS-135. Secretory granules (SG). (C) Quantitative TEM immunogold labeling of secretory proteins in PG of mice flown on STS-131 and STS-135, and the Bion-M1 biosatellite. α-Amylase, parotid secretory protein (PSP), PRP and the type II regulatory subunit of protein kinase A (RII) are present in acinar cell secretory granules; demilune cell and parotid protein (DCPP) is present in intercalated duct cell secretory granules. Labeling results are shown as a percentage of the corresponding habitat control mice ± SEM.* 

PKA-RII also is present in the nucleus, where it is involved in regulating several genes [31, 32]. No significant differences were seen in nuclear PKA-RII in parotid

Compared to their habitat controls, expression of α-amylase, a digestive enzyme, was decreased in the parotid glands of mice flown on the space shuttles, but increased in mice flown on the Bion-M1 biosatellite. Parotid secretory protein (PSP), an antimicrobial protein [25, 26], was slightly increased in mice from the Bion-M1 flight. Proline-rich protein (PRP), a calcium and polyphenol binding protein [27, 28], was decreased in mice from STS-131, increased in mice from STS-135, and decreased, but not significantly, in mice from the Bion-M1 flight. The type II regulatory subunit of protein kinase A (PKA-RII), a stress marker secreted into saliva [29], was slightly decreased in mice from the 3 flights. Demilune cell and parotid protein (DCPP), secreted by intercalated duct cells and believed to have antimicrobial activity [30], was increased in mice from the STS-131 flight, but was

several salivary proteins following spaceflight [12, 13] (**Figure 5**).

unchanged in mice from STS-135 and Bion-M1 flights.

acinar cells of mice from the 3 flights (**Figure 6**).

*Beyond LEO - Human Health Issues for Deep Space Exploration*

The underlying basis for these changes is believed at least in part to be the loss of neural stimulation due to reduced masticatory activity. This is indicated by experiments where withholding food for 24–48 h, or feeding rats a liquid diet, results in numerous autophagic vacuoles containing degenerating secretory granules and an

*TEMs of PG from STS-135 flight mice. (A) An acinar cell contains 2 autophagic vacuoles (AV), and a macrophage (Mac) has engulfed an apoptotic acinar cell (APOP). (B) Intercalated duct cells contain apical vacuoles (arrows)* 

*resulting from endocytosis of secretory proteins from the lumen (L) (with permission, from [13]).*

The mice from the Bion-M1 mission all gained weight during the flight but were fed a soft paste diet. The mice from the space shuttle missions had *ad libitum* access to NASA rodent food bars [18], but lost a small amount of weight, as did the habitat control mice (maintained on the ground for the same length of time in the same habitats as the flight mice). This suggests that the mice on the space shuttle flights may not have eaten the amount of food sufficient to maintain their body weight. Since the number of autophagic vacuoles and frequency of apoptosis was greater in flight mice than in the habitat controls, microgravity also appears to affect these processes. Endocytosis of salivary proteins by duct cells occurs in experimental diabetes [19, 20]. This suggests that the proteins may have an altered structure that is recognized as foreign by the duct cells; these cells are capable of endocytosing foreign proteins introduced in a retrograde fashion *via* the main excretory duct [21–23]. Alternatively, duct cell function in the flight mice may have been altered.

In contrast to the PG, no morphological changes were evident in the SMG or SLG of the flight mice. Similarly, feeding a liquid diet has no apparent effect on the

Relative changes in secretory protein expression can be analyzed by electron microscopic immunogold labeling. Determination of the labeling density (gold particles/μm2

**2.2 Salivary protein expression studied using two major approaches**

increase in the number of apoptotic cells in the PG [14–17].

morphology of the rat SMG and SLG [24].

*2.2.1 Electron microscopic immunogold labeling*

**58**

**Figure 4.**

#### **Figure 5.**

*TEM immunogold labeling for proline-rich protein (PRP) in PG acinar cells of (A) flight and (B) habitat ground control mice from STS-135. Secretory granules (SG). (C) Quantitative TEM immunogold labeling of secretory proteins in PG of mice flown on STS-131 and STS-135, and the Bion-M1 biosatellite. α-Amylase, parotid secretory protein (PSP), PRP and the type II regulatory subunit of protein kinase A (RII) are present in acinar cell secretory granules; demilune cell and parotid protein (DCPP) is present in intercalated duct cell secretory granules. Labeling results are shown as a percentage of the corresponding habitat control mice ± SEM. +, p = 0.065; \*, p < 0.05; #, p < 0.01.*

of secretory granules of PG acinar and duct cells showed alterations in the expression of several salivary proteins following spaceflight [12, 13] (**Figure 5**).

Compared to their habitat controls, expression of α-amylase, a digestive enzyme, was decreased in the parotid glands of mice flown on the space shuttles, but increased in mice flown on the Bion-M1 biosatellite. Parotid secretory protein (PSP), an antimicrobial protein [25, 26], was slightly increased in mice from the Bion-M1 flight. Proline-rich protein (PRP), a calcium and polyphenol binding protein [27, 28], was decreased in mice from STS-131, increased in mice from STS-135, and decreased, but not significantly, in mice from the Bion-M1 flight. The type II regulatory subunit of protein kinase A (PKA-RII), a stress marker secreted into saliva [29], was slightly decreased in mice from the 3 flights. Demilune cell and parotid protein (DCPP), secreted by intercalated duct cells and believed to have antimicrobial activity [30], was increased in mice from the STS-131 flight, but was unchanged in mice from STS-135 and Bion-M1 flights.

PKA-RII also is present in the nucleus, where it is involved in regulating several genes [31, 32]. No significant differences were seen in nuclear PKA-RII in parotid acinar cells of mice from the 3 flights (**Figure 6**).

)

#### **Figure 6.**

*Quantitative TEM immunogold labeling of nuclear PKA-RII in PG of mice flown on STS-131 and STS-135, and the Bion-M1 biosatellite. Labeling results are shown as a percentage of the corresponding habitat control mice ± SEM.*


#### **Table 1.**

*Quantitative TEM immunogold labeling of secretory proteins in SMG of female mice flown on STS-131 and STS-135, and male mice flown on the Bion-M1 biosatellite.*

The expression of SMG secretory proteins was essentially unchanged in mice from the 13–15-day space shuttle flights. However, mice from the Bion-M1 flight showed significantly increased expression of an acinar cell protein, salivary androgen binding protein alpha (SABPα), a pheromone involved in mate selection [33], and the granular convoluted duct cell proteins epidermal growth factor (EGF) and nerve growth factor (NGF) (**Table 1**). PRP, present in acinar cell secretory granules of both sexes, and submandibular gland protein C (SMGC), present in terminal tubule cell secretory granules of female mice, were not significantly different from controls. In the SLG, PSP expression by demilune cells was significantly increased in mice from the space shuttle STS-131 flight, but not in mice from STS-135 [34]. The expression of both PKA-RII in demilune cells, and the acinar cell mucin Muc19, were increased, but not significantly.
