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DOI: 10.1080/01926230601042494
Nasal Cytotoxic and Carcinogenic Activities of Systemically Distributed Organic ChemicalsNew York Medical College, Department of Pathology, Valhalla, New York 10595, USA Correspondence: Address correspondence to Gary M. Williams, Department of Pathology New York Medical College, Valhalla, NY 10595; e-mail: GaryWilliams{at}nymc.edu
Toxicity and carcinogenicity in the mucosa of the nasal passages in rodents has been produced by a variety of organic chemicals which are systemically distributed. In this review, 14 such chemicals or classes were identified that produced rodent nasal cytotoxicity, but not carcinogenicity, and 11 were identified that produced nasal carcinogenicity. Most chemicals that affect the nasal mucosa were either concentrated in that tissue or readily activated there, or both. All chemicals with effects in the nasal mucosa that were DNA-reactive, were also carcinogenic, if adequately tested. None of the rodent nasal cytotoxins has been identified as a human systemic nasal toxin. This may reflect the lesser biotransformation activity of human nasal mucosa compared to rodent and the much lower levels of human exposures. None of the rodent carcinogens lacking DNA reactivity has been identified as a nasal carcinogen or other cancer hazard to humans. Some DNA-reactive rodent carcinogens that affect the nasal mucosa, as well as other tissues, have been associated with cancer at various sites in humans, but not the nasal cavity. Thus, findings in only the rodent nasal mucosa do not necessarily predict either a toxic or carcinogenic hazard to that tissue in humans.
Key Words: Nose • cancer • toxicity • DNA adducts • mutagens • metabolism • safety assessment Abbreviations: 2,6-DMA, 2,6-dimethylaniline • 3-MI, 3-methylindole • ADCP, 3,5-aminodichloropyridine • APAP, acetaminophen • BGs, Bowman’s glands • CYP(s), cytochrome P450(s) • DCB(s), dichlorobenzene(s) • DCBN, 2,6-dichlorobenzonitrile • GSH, glutathione (reduced) • HMPA, hexamethylphosphoramide • IARC, International Agency for Research on Cancer • IDPN, β,β'-iminodipropionitrile • NM, nasal mucosa • NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone • NPIP, N-nitrosopiperidine • NTP, National Toxicology Program • OE, olfactory epithelium • OM, olfactory mucosa • PA, phenacetin • PCB(s), polychlorinated biphenyl(s) • PDR, Physicians’ Desk Reference • RE, respiratory epithelium, ciliated pseudostratified • SD, Sprague
The nasal passages of rodents are increasingly recognized as an important target site of action for certain toxins and carcinogens. Up through 1997, excellent reviews of xenobiotic effects in rodent nasal mucosa (NM) were provided by several investigators (Dahl and Hadley, 1991; Brittebo, 1997; Monticello and Morgan, 1997; Schuller, 1997). The need for an understanding of carcinogenic susceptibility factors in rodent and human nasal tissues in order to develop plausible modes of action has been highlighted (Bogdanffy et al., 1997). The purpose of this review is to expand on these earlier reports with new information on this topic directed to evaluation of human risk from systemic exposure to rodent nasal cytotoxins and carcinogens. After acceptance of this manuscript, two other relevant papers were published (Harkema et al., 2006; Genter, 2006). For each chemical identified as producing either nasal cytotoxicity or carcinogenicity, in this review, authoritative sources were accessed and literature searches were conducted to identify human effects.
In animals and humans, the elaborate nasal passages are covered by the NM, which is composed of several types of epithelium. The most anterior portion, the nasal vestibule, is covered by stratified squamous epithelium. Posterior to the nasal vestibule, the epithelium is composed of nonciliated cuboidal transitional epithelium. Immediately posterior to the transitional epithelium, the epithelium is ciliated and assumes the structure of pseudostratified respiratory epithelium (RE). Farthest posterior, in the craniodorso-posterior aspect of the nasal cavity, the region designated as the olfactory mucosa (OM), is lined with specialized olfactory epithelium (OE). The complex OE consists of 3 layers of cells: (i) a lower basal cell compartment; (ii) a receptor or sensory cell compartment containing the olfactory (sensory) neurons, their precursor basal cells, together with the cells of Bowman’s glands (BGs) and ducts; and (iii) a supporting (or sustentacular) cell compartment (Harkema, 1990, 1991; Legrier et al., 2001). The subepithelial BGs within the lamina propria of the OE constitute exocrine glands whose excretions are passed through excretory ducts to the surface of the OE to moisten the mucosal surface (Uraih and Maronpot, 1990; Harkema, 1991). The main anatomical difference in the NM across animal species and humans is in the percentage of OE lining the NM surface; the OE occupies 50% of the surface in rodents, but only 10% in humans (Harkema, 1990), reflecting the high reliance of rodents on the sense of olfaction. Conversely, the transitional epithelium occupies only 10% and the RE 35% of the NM surface in rats and 25% and 60% respectively, in humans (Reznik, 1983; Uraih and Maronpot, 1990; Harkema, 1990, 1991; Morgan et al., 1991; Mery et al., 1994; Menco and Morrison, 2003). As described here, the OE is a site of toxicity of many xenobiotics. The receptor neurons of the OE are bipolar cells whose dendrites extend to the epithelial surface and whose axons connect to the olfactory bulb. The cilia of the olfactory neurons form dendritic knobs that contain odorant receptors (Zhang and Firestein, 2002). The olfactory neurons have the unusual ability for neurons to periodically replace themselves (Menco, 1983; Harkema, 1990; Reznik, 1990; Uraih and Maronpot, 1990; Menco and Morrison, 2003). At birth the basal cell progenitors of the olfactory neurons in mice exhibit high mitotic activity that diminishes significantly by 3 months after birth and remains at a lower level throughout life (Legrier et al., 2001). In the rat, the life span of olfactory neurons is approximately 20–28 days (Uraih and Maronpot, 1990; Legrier et al., 2001), while in the mouse, the life span of some neurons is 1 year (Hinds et al., 1984). The entire OE has a high level of cell turnover (Moulton, 1974) with OE cells being renewed by their progenitors, the globose basal cells (Moulton, 1974; Schwartz-Levey et al., 1991; Caggiano et al., 1994; Huard and Schwob, 1995). The rate of replacement of the OE has been suggested to reflect injury by airborne toxins (Farbman, 1990; Calderon-Garciduenas et al., 1998). The rodent NM possesses substantial chemical biotrans-formation capability (Hadley and Dahl, 1982; Dahl, 1985; Longo et al., 1988; Dahl and Hadley, 1991; Bereziat et al., 1995; Genter et al., 1995b; Thornton-Manning and Dahl, 1997; Genter, 2004), which may play a role in olfaction (Anholt, 1989; Nef et al., 1989; Carr et al., 1990). Species differ in their specific levels of biotransformation capability (Hadley and Dahl, 1983; Thornton-Manning and Dahl, 1997). Relatively little is known about NM biotransformation activity in humans, but biotransformation enzymes are definitely present (Gervasi et al., 1991; Lewis et al., 1994; Yokose et al., 1999; Zhang et al., 2005). Comparisons of rodent and human nasal tissues generally reveal the former to have higher biotransformation activities (Feng et al., 1990; Bogdanffy et al., 1998; Heydens et al., 1999). In the rat NM, the ethmoid turbinates, which are a frequent site for tumor formation, have very high chemical biotransformation activity compared to other species (Hadley and Dahl, 1982; Hadley and Dahl, 1983; Dahl, 1985; Longo et al., 1988; Sabourin et al., 1988). In particular, in rodents, the phase I oxidation enzymes, the cytochromes P450 (CYPs) are present at high levels in the NM, being mainly expressed in the RE and OE (Brittebo, 1997; Thornton-Manning and Dahl, 1997; Ling et al., 2004). Indeed, the most highly expressed genes in the NM are those of Phase I and II biotransformation enzymes (Hester et al., 2002). The NM CYPs include 1A1, 2A, 2C, 2E1, 2G1, 3A, and 4B (Brittebo, 1997; Thornton-Manning and Dahl, 1997; Gu et al., 1999; Wang et al., 2002). The CYP2A subfamily enzymes are involved in the biotransformation of many xenobiotics. In the rat, CYP2A3 is expressed in the NM (Robottom-Ferreira et al., 2003), whereas in mouse NM CYP2A5 is present (Piras et al., 2003) and CYP2A6 in humans (Liu et al., 1996). Among the CYPs, CYP2G1 activity (Nef et al., 1990; Hua et al., 1997) or transcripts (Yu et al., 2005) are expressed in the OE of mice, rats, and rabbits (Ling et al., 2004), but in humans the gene has multiple mutations and there is no functional enzyme activity (Sheng et al., 2000). In one study, human nasal tissue had only 0.15% of the CYP (7-ethoxycoumarin-O-deethylase) activity of that of the rat (Feng et al., 1990). Another family of oxidation enzymes, flavin-containing monooxygenases, is present at higher levels in the rat OM than in the liver (Genter and Ali, 1998). Phase II conjugating enzymes such as glutathione-S-transferase and glucuronyl transferase are also present in the NM (Thornton-Manning and Dahl, 1997). N-acetyltransferase transcripts, specifically the N-acetyltransferase 1 enzyme, which is widely distributed in the body, is present in the RE and OE, as well as the BGs (Debiec-Rychter et al., 1996). In fact, activity in the OM of Long–Evans rats is markedly higher than in the liver (Genter, 2004). Other enzymes, such as epoxide hydrolase, are higher in rats when compared to other species such as mice (Green et al., 2001). The activities of some of the epoxide hydrolases are similar to those of liver with respect to polycyclic aromatic hydrocarbon biotransformation (Bond, 1983). These hydrolases often act to detoxify reactive arene oxides, but in other instances they can be involved in multistep activation of some aromatic hydrocarbons (Levin et al., 1980). In contrast to the situation with epoxide hydrolase, carboxylesterase activity is reported to be slightly higher in mice than in rats (Stott and McKenna, 1985). In both species, activity was greater in the OE than in the RE (Bogdanffy et al., 1987; Frederick et al., 1994), being present in the OE in sustentacular cells and BGs (Robinson et al., 2002). Considering the proficiency of the rodent NM in chemical biotransformation, this tissue might reasonably be regarded as a veritable second liver. Moreover, like the liver, it is positioned at a portal of entry for xenobiotics. Several of the enzyme systems present in the NM are known to be inducible (Bond, 1983; Gillner et al., 1987; Longo and Ingelman-Sundberg, 1993; Bereziat et al., 1995; Nikula et al., 1995; Thornton-Manning and Dahl, 1997). However, the hepatic CYP2A5 inducers pyrazole and phenobarbital did not affect message or protein expression in mouse OM (Piras et al., 2003) and 3-methylcholanthrene and pyrazole did not induce CYP2A3 in rat NM (Robottom-Ferreira et al., 2003). Antioxidant enzymes, including superoxide dismutase, catalase, glutathione peroxidase and DT-diaphorase were reported to be at higher levels in rat NM than in the lung of rats (Reed et al., 2003). Enzymes were higher in the RE than OE.
The NM also possesses cellular protective systems, including the antioxidants ascorbate and As a consequence of species differences in enzymatic activities, the effect of a specific chemical in the NM may differ between species depending upon the specific pathway involved in the biotransformation of the compound. Where such information was found for a specific chemical, it was included in this review. However, broad generalizations about susceptibility of NM to chemical toxicity are not possible. Uptake of chemicals into systemic circulation through the NM is well recognized, and nasal administration is used as a delivery route for several drugs (Hussain, 1998). Additionally, transport of chemicals from the OE to the olfactory bulb takes place along neuronal axons (Shipley, 1985; Hastings and Evans, 1991; Tjälve et al., 1996).
In spite of the dynamic condition of the NM, spontaneous nasal tumors are rare in rodents. In the Fischer 344 rat, which is used in National Toxicology Program (NTP) bioassays, the spontaneous nasal tumor incidence is usually <0.5% (Rao et al., 1990; Haseman and Clark, 1990; Haseman and Hailey, 1997) and similar to that seen in the Wistar rat (Feron et al., 1990). B6C3F1 mice, as reported in the NTP database, have an even lower incidence (Haseman and Elwell, 1996). The majority of spontaneous nasal tumors are squamous cell carcinomas (Feron et al., 1990; Haseman and Clark, 1990; Rao et al., 1990; Haseman and Elwell, 1996; Haseman and Hailey, 1997), which arise in the nares. The higher background of NM tumors in the rat implicates a genetic susceptibility, which may result from a variety of causes including differences in biotransformation activities or cell proliferation, and probably contributes to the greater inducibility of such tumors in the rat. A variety of chemicals, administered orally or perenterally, has been found to produce nasal cytotoxicity (Table 1) and some to induce tumors of the rodent nasal cavity (Table 2). Chemicals that have carcinogenic activity to the anterior squamous epithelium and are active upon direct contact with the NM when administered by inhalation (Feron et al., 2001), such as formaldehyde, are not detailed in this review, although inhalation is an important route of human exposure and many volatile carcinogens act directly on the NM. Also, some chemicals that are cytotoxic to the NM by the inhalation route produce similar effects when delivered systemically (Keller et al., 1997; Lee et al., 2005). Nevertheless, here we focus on organic chemicals that have cytotoxic and carcinogenic activity in the NM upon systemic distribution following oral or perenteral administration.
Many, but not all, such nasal cytotoxins have carcinogenic activity. Among chemicals with carcinogenic activity for the RE and OE, some are clearly DNA reactive, whereas others have not been shown to bind to DNA, but were reported to bind to protein and produce cytotoxicity. Cytotoxicity and reparative compensatory cell proliferation may be important contributors to carcinogenic activity in the NM, even with DNA-reactive agents, as shown for a variety of tissues (Butterworth et al., 1992; Cunningham, 2000; Williams et al., 2000). Many of the organic chemicals that are cytotoxic or have carcinogenic activity in the NM with systemic distribution are single ring compounds or liberate single ring components following biotransformation. Most such chemicals produce greater effects in the NM of the rat than in the mouse and males are often more susceptible than females. The organ specificity of these carcinogens appears in part to result from high and sometimes specific biotransformation within the RE and OE (Hadley and Dahl, 1983; Dahl and Hadley, 1991). The characteristics of these agents are reviewed herein and the relevance of their effects in experimental animals to human cancer risk assessment is discussed.
At least 14 chemicals or classes of compounds reviewed in this section have produced nasal cytotoxicity, but either have not produced nasal tumors or have not been tested for systemic carcinogenicity but are deemed unlikely to be nasal carcinogens by that route. Acetaminophen (APAP) or paracetamol (Figure 1) is a widely used analgesic and is a biotransformant of phenacetin (Figure 30), which is carcinogenic to the NM (see below). With oral doses of 600 mg/kg bw ip, APAP was reported to be toxic to the NM of mice (Hart et al., 1995; Genter et al., 1998). The toxicity in the mouse NM is not dependent upon hepatic biotransformation (Gu et al., 2004), but rather involves local bioactivation to a N-acetyl-p-benzoquinoneimine (Miner and Kissinger, 1979), the chemically reactive species which binds to protein (Muldrew et al., 2002) (Figure 1), mediated by CYPs 2E1, 2A5 and 2G1 (Hinson, 1983; Gu et al.,1998; Chen et al., 1998). APAP was not genotoxic in most systems (International Agency for Research on Cancer, 1999c). Whereas APAP was reported to bind to mouse liver and kidney DNA (Rogers et al., 1997), no binding was reported in rat liver (Hasegawa et al., 1988) or rat liver, colon or bladder (Williams et al., 2002). In oral carcinogenicity studies of APAP in mice and rats at doses up to 6000 ppm in the diet, equivocal effects in female rats were reported for some tissues as assessed by the International Agency for Research on Cancer (International Agency for Research on Cancer,1999c), but no nasal tumors were observed (Hiraga and Fujii, 1985; National Toxicology Program, 1993a). In a review of about a dozen epidemiological studies, APAP use was not noted to be associated with any nasal toxicity and was not considered to be carcinogenic in humans (International Agency for Research on Cancer, 1999c). Recently, however, heavy use has been reported to be associated with a slight increase in renal cancer (Kaye et al., 2001).
Antimicrotubule Agents Vincristine sulfate, vinblastine sulfate, vindesine sulfate (Figure 2), and paclitaxel (formally called taxol) (Figure 3) at doses of ~2–12 mg/kg bw administered intravenously produced apoptosis in the OE of BALB/c mice by 24 hours (Kai et al.,2002, 2004, 2005). This progressed to atrophy by 15 days. Whole-body radio-luminography revealed concentrations of vincristine sulfate in nasal tissues about 2-fold higher than that in blood (Kai et al., 2005).
In limited studies, evidence was not found for carcinogenicity of vinblastine sulfate (International Agency for Research on Cancer, 1987h) or vincristine sulfate (International Agency for Research on Cancer, 1987i). In spite of use as anticancer medicines, no information was found on human nasal cytotoxicity or carcinogenicity. Aryl methyl sulfones (Figure 4) are biotransformants formed from chloro aryl hydrocarbons derived from compounds such as 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) and polychlorinated biphenyls (PCBs) following conjugation with glutathione (GSH) and degradation via mercapturic acid derivatives. 2,6-dichlorophenyl methylsulfone, as a single ip dose, produced necrosis preferentially in the BG and neuroepithelium in the dorsomedial olfactory region (Franzén et al., 2003), probably mediated by CYP2A5 activation, one of the prominent CYPs in the mouse (Zhuo et al., 2004; Franzén et al., 2006). By contrast, only minor damage occurred at this site in rats dosed with the 2,5-chlorinated isomer.
Assessments of nasal cytotoxicity or carcinogenicity to humans have not been reported. Benzophenone (Figure 5) occurs naturally and is used the manufacture of a wide variety of industrial chemicals. In a 14-week study, mice receiving up to 20,000 ppm benzophenone in the diet exhibited no changes in the NM. With administration for 2 years of doses of 1200 ppm in the diet, male and female mice developed metaplasia of the OE, but rats were not affected (National Toxicology Program, 2006).
In these carcinogenicity studies, benzophenone produced increases in male mouse liver neoplasms and male rat kidney neoplasms, likely through epigenetic mechanisms, but tumors were not produced in the NM. Assessments of nasal cytotoxicity or carcinogenicity to humans were not found (Hazardous Substances Data Bank, 2006). Bromobenzene (Figure 6) is used in organic synthesis, especially to make phenyl magnesium bromide; as an additive to motor oils; as a solvent, especially for crystallizations on a large scale and where a heavy liquid is desirable. It elicited nasal toxicity when administered ip at > 4.8 mmol/kg body weight and showed high levels of tissue binding particularly to the BGs (Brittebo et al., 1990). Human exposures have occurred in occupational situations and at low levels (normally <1 ppb) in drinking water (Environmental Working Group and EWG Action Fund, 2006). The U.S. Environmental Protection Agency estimates that exposures of children to 4000 ppb for 10 days is not expected to cause any adverse, noncarcinogenic effects. However, in February, 2005, the U.S. Environmental Protection Agency included bromobenzene in the Drinking Water Contaminant Candidate List 2 (US EPA, 2005). While bromobenzene is highly toxic, there is no evidence for carcinogenicity to the NM or to any other site (National Research Council, 2006).
Chloroform (CHCl3) (Figure 7) has been used as a solvent and is generated as a trace contaminant in the chlorination of drinking water with a maximum allowable concentration as total trihalomethanes of 80 ppb. Oral administration to female F344 rats of 34 mg/kg/day or greater produced degeneration of the OE and superficial BGs (Larson et al., 1995), which was not associated with detectable olfaction deficit (Dorman et al., 1997). The OE, RE, and BGs have substantial chloroform biotransformation capabilities (Lofberg and Tjalve, 1986; Constan et al., 1999), leading to generation of phosgene, which in turn can react with proteins (Fabrizi et al., 2003) (Figure 7). Chloroform administration produced liver and kidney tumors in mice and kidney tumors in rats, but no nasal tumors (International Agency for Research on Cancer, 1999a). Also, chloroform did not produce nasal tumors in sensitive TP53+/– mice (Storer et al., 2001). IARC (International Agency for Research on Cancer, 1999a) concluded that in humans the evidence for carcinogenicity of chloroform was inadequate. No indication of any effect on the nasal passages was found (Hazardous Substances Data Bank, 2006).
Coumarin (Figure 8) is a naturally occurring fragrant compound that has been used in consumer products and as a food additive. A single ip injection of 50 mg/kg bw to Wistar rats and C57BL/6 mice produced reductions at 48 hours in CYP2A and CYP2G, the major CYPs in mice (Zhuo et al., 2004), in the OE, but not in the liver (Gu et al., 1997). By 12 hours, necrosis of the OE and underlying BG was evident and this progressed up to 48 hours. The principal biotransformants resulted from 3- or 7-hydroxylation or formation of a transient 3,4-epoxide which, rather than rearranging to form 3-hydroxycoumarin, yields o-hydroxyphenylacetaldehyde, which can undergo further oxidation to the corresponding acetic acid derivative (Born et al., 2002) (Figure 8). In the mouse, OM-specific CYP2G1 is the major enzyme responsible for biotransformation, producing 7-hydroxycoumarin and o-hydroxyphenylacetaldehyde as the predominant products (Zhuo et al., 1999). Other CYPs were also active.
Although there is some evidence for induction of various neoplasms in rodents by coumarin, no tumoriginicity has been reported in the nasal cavity (National Toxicology Program, 1993b). Coumarin has not been reported to be associated with nasal toxicity or carcinogenicity to humans. Dichlorobenzenes (substituted). 2,6-Dichlorobenzonitrile (DCBN) (dichlorobenil) and 2,6-dichlorothiobenzamide (chlorthiamid) are dichlorobenzene (DCB) derivatives (Figure 9), which have been used as herbicides.
In mice, single ip injections of DCBN (12, 25, 50 mg/kg) produced necrosis in the dorsomedial OE (Brandt et al., 1990). DCBN (25 mg/kg) and chlorthiamid (12 mg/kg) were toxic to the OE of mice with oral administration (Eriksson and Brittebo, 1995b; Mancuso et al., 1997). The injury produced by acute doses of DCBN persisted for up to 6 months. DCBN bound specifically to the epithelium of mouse BGs (Eriksson and Brittebo, 1995a). Subsequently, methylsulfonyl-2,5-DCB and methylsulfonyl-2,6-DCB were reported to be localized in the OM of female NMRI mice, but only the latter was toxic to the OE (Bahrami et al., 1999). The parent compounds, 1,3- and 1,4-DCBs, and 1,2,3-trichlorobenzene, were nontoxic to mouse OM (Bahrami et al., 1999). However, 1,4-DCB produced epithelial necrosis of the nasal turbinates in male and female rats when given by gavage for 13 weeks at 1,200 and 1,500 mg/kg (National Toxicology Program, 1987). In a study of a wide range of 2,6-DCBs, 2,6-dichloronitrobenzene and dichlorobenzaldehyde were identified as having OM toxicity, but a simple explanation of nasal toxicity based on structure activity relationships could not be identified (Carlsson et al., 2004). CYP activity of the NM underlies the toxicities of these compounds, mediating selective covalent binding of the toxicants to the NM, especially the dorsal medial meatus (Brittebo, 1997). CYP 2A5 and CYP 2G1 activate DCBN to a reactive intermediate (Brandt et al., 1990), which may be a 2,3-arene oxide (Genter et al., 1995b; Ding et al., 1996). The region of injury lacks microsomal epoxide hydroxylase (Genter et al., 1995b), which suggests an inability to detoxify an epoxide. DCBN induced liver tumors in rats and several types of tumors in mice, including liver tumors, but there was no indication of nasal tumors (Cox, 1997). 1,4-DCB produced kidney tumors in rats, but no nasal tumors (National Toxicology Program, 1987). None of these DCBs has been reported to be associated with nasal toxicity or carcinogenicity in humans.
Aliphatic Nitriles
The OM injury produced by IDPN results in degeneration of nerve terminals in the glomeruli of the olfactory bulb (Boadas-Vaello et al., 2005). Two other nitriles, allylnitrile and cis-crotononitrile, when given orally to rats on 3 consecutive days produced degradation in the olfactory bulbs indicative of injury to the OM (Boadas-Vaello et al., 2005). Reports of experimental carcinogenicity of IDPN or information on human nasal cytotoxicity or carcinogenicity were not found. Methylacrylonitrile (Figure 10) is an unsaturated alkyl nitrile that is widely used in the preparation of homopolymers and copolymers. When administered by gavage to F344 rats, the dose of 30 mg/kg bw given for 2 years, produced atrophy and metaplasia of the OE in bothmales and females, but no neoplasms (Nyska and Ghanayem, 2003). No information was found on human nasal cytotoxicity or carcinogenicity (Hazardous Substances Data Bank, 2006). Methimazole (1-methylimidazole-2-thiol) (Figure 11) is a nitrogen heterocyclic medicine that has been used for the treatment of hyperthyroidism. In Long–Evans rats, administration of 25 mg/kg by ip injection or 50 mg/kg by intragastric instillation as single doses with animals sacrificed 32 hours later, caused almost complete destruction of the OE (Genter et al., 1995a). Methimazole was also toxic to the OE in mice (Bergman and Brittebo, 1999); in NMRI mice, 2 doses of 50 mg given by ip injection 3 days apart produced damage to the OE and BGs (Bergman et al., 2002). The damage was rapidly repaired with only minor changes 3 months later (Bergman et al., 2002). Methimazole showed selective covalent binding in the BGs as well as the bronchial epithelium in the lungs and centrilobular regions of the liver following an intravenous (iv) injection in mice (Bergman and Brittebo, 1999). The related chemicals, 2-methylimidazole and 4-methylimidazole, did not produce nasal toxicity but induced thyroid lesions (Chan et al., 2006), as does methimazole. Methimazole also produced a transient loss of the sense of smell in rats (Genter et al.,1996; Xu and Slotnick, 1999), as does carbimazole (Genter, 1998), a carbethoxy derivative of methimazole, which is converted in the body to methimiazole (Wishart et al., 2006).
Pretreatment of mice with the CYP inhibitor metyrapone completely abolished covalent binding of methimazole to the OE and bulb (Bergman and Brittebo, 1999), indicating a requirement for biotransformation. Methimazole is probably bioactivated to an S-oxide (Genter et al., 1995a) by a flavin-containing monooxygenase, an enzyme abundant in the NM (Genter and Ali, 1998). Methimazole produced rapid depletion of nonprotein sulfhydral groups, mainly GSH, in the OE of mice (Bergstrom et al., 2003). Methimazole is a nitrogen heterocyclic, but it seems from analog studies that oxidation of the exocyclic thiol is critical for nasal toxicity (Genter et al., 1995a), although this may not provide a complete explanation and ring epoxidation and subsequent cleavage may also play a role (Mizutani et al., 2000). In a carcinogenicity study in rats, neoplasms were increased only in the thyroid gland (Lilly, 1996), while a study in mice yielded no increase in neoplasia (Jemec, 1970). The development of the thyroid tumors in rats is likely as a result of the goitrogenic effects of the drug (Capen, 1994). No cytotoxicity to human NM has been noted (Bartalena et al., 1996), although loss of the sense of smell has been reported (Schiffman and Gatlin, 1993), as also with exposure to carbimazole (Erikssen et al., 1975; Neundorfer, 1987), similarly to rats (Genter et al., 1996; Genter, 1998). Detailed studies of possible OM degeneration in humans have not been undertaken. No information was found on human carcinogenicity. 3-Methylindole (3-MI) (Figure 12) is produced from tryptophan by fermentation in the intestinal tract (Wiethoff et al., 2001) and is present in cigarette smoke (Hoffmann and Rathkamp, 1970). Intraperitoneal injection (ip) of 400 mg/kg to C57BL mice produced OE necrosis (Turk et al., 1986, 1987), which resulted in olfaction deficits (Peele et al., 1991; Miller and O’Bryan, 2003). The BGs and sustentacular cells were predominantly affected (Miller and O’Bryan, 2003). 3-MI is biotransformed by lung CYPs to form free radicals (Bray and Kubow, 1985).
Bioactivated 3-MI formed adducts with isolated or cellular DNA (Regal et al., 2001). Its reactive intermediate, 3-methyleneindolenine (Figure 12) was generated by CYP2F (Wang et al., 1998) and bound to DNA, whereas an epoxide may be involved in reaction with softer nucleophiles such as glutathione and proteins (Skordos et al., 1998; Lanza et al., 1999). Administration of 3-MI to rats and rabbits produced protein thioether adducts in lung, kidney, and liver, as demonstrated by an enzyme-linked immunosorbent assay (Kaster and Yost, 1997), but NM was not studied. The specific contribution of either or both of the above reactive products to nasal toxicity in mice is not resolved. No report of 3-MI carcinogenicity testing was found and nasal cytotoxicity or carcinogenicity related to 3-MI exposure in humans has not been reported. Naphthalene (Figure 13), the next higher homolog of benzene (Figure 19), is a major constituent of coal tar and creosote, and is formed in most incomplete combustion processes. Its main use is in the production of phthalic anhydride, while its use as a moth repellant is declining. Naphthalene is quite volatile and most human exposures probably occur via inhalation. When administered ip at 200 mg/kg bw to SD rats, it produced severe injury throughout the olfactory region (Lee et al., 2005). Naphthalene was tested systemically for carcinogenicity by oral administration in one study in rats, by intraperitoneal administration in newborn mice and in rats and by other routes. All these studies were considered too limited for an evaluation of the experimental carcinogenicity of naphthalene by IARC (International Agency for Research on Cancer, 2002), which considered it a possible human carcinogen. Subsequently, induction of olfactory neuroblastomas and respiratory epithelial adenomas by inhalation exposure was reported (Long et al., 2003). The lack of clear carcinogenicity to the NM by oral or perenteral routes may reflect systemic detoxification. No report of nasal cytotoxicity or carcinogenicity in humans was found (Hazardous Substances Data Bank, 2006).
Polychlorinated biphenyls (PCBs) (Figure 14) Among dioxin-like compounds tested for carcinogenicity, the PCB binary mixtures of either 3,3',4,4',5-pentachlorobiphenyl (PCB 126) and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153) or PCB 126 and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 118) produced nasal lesions in female SD rats (Nyska et al., 2005). Inflamation occurred with PCB 126 and PCB 153 at doses of 300 µg/kg bw, while RE hyperplasia occurred only at the highest dose of 1000 µg/kg bw. Although PCBs induce liver tumors in rodents, induction of nasal neoplasms has not been reported (International Agency for Research on Cancer, 1987f).
Up to 1987, no report of nasal cytotoxicity or carcinogenicity related to PCB exposure in humans was available (International Agency for Research on Cancer, 1987f) and no subsequent reports were found. 3-Trifluoromethylpyridine (Figure 15) is a starting material for the synthesis of herbicides. With inhalation, it produced nasal toxicity in rats (Gaskell et al., 1990), and, when given orally, accumulated in the ethmoid turbinates and dorsal meatus of the nasal passages and bound to proteins (Hext and Lock, 1992), suggesting that it would be cytotoxic with systemic distribution. The structurally related aminodichloropyridine is cytotoxic and carcinogenic to the NM with systemic distribution (see below), but no report of 3-trifluoromethylpyridine carcinogenicity testing or human toxicity was found.
Overview of Cytotoxicity Of the 14 compounds or classes of compounds discussed here and summarized in Table 1, which produced nasal cytotoxicity with oral or parenteral administration, none produced nasal neoplasms, although several increased tumor incidence at other sites, namely benzophenone, chloroform, coumarin, DCBN, methylindole and PCB. However, the conditions for induction of nasal tumors may not have been met, and a detailed search for nasal tumors may not always have been performed, especially in earlier studies. In fact, it is possible that with appropriate systemic testing, naphthalene might be a nasal carcinogen. In humans, no nasal cytotoxicity or carcinogenicity was found.
Mechanisms of Rodent Nasal Toxicity Where appropriate investigations were conducted, many of the chemicals bound selectively to the NM, which, no doubt, underlies their nasal cytotoxicity. Perhaps, gene expression studies (Hester et al., 2002, 2005) would shed some light on tissue responses that may differ between agents lacking nasal carcinogenicity and those that are carcinogenic to the NM (discussed below).
Human Effects of Rodent Nasal Cytotoxins
The principal chemicals reviewed here all have exerted some degree of carcinogenic activity, as well as cytotoxicity, in the rodent NM under certain conditions of oral or perenteral administration.
Aniline Derivatives
p-Cresidine was also carcinogenic in B6C3F1 mice, causing carcinomas of the urinary bladder in both genders and hepatocellular carcinomas in females, but not nasal tumors (National Toxicology Program, 1979b). p-Cresidine is used as the positive control in the TP53+/– transgenic mouse bioassay in which it produces bladder neoplasms in both genders and hepatocellular carcinomas in females, but no nasal tumors (Storer et al., 2001), although NM atrophy, necrosis, and degeneration have been reported in DNA repair-deficient C57BL/6XPA–/– mice (Mortensen et al., 2002). These findings in the mouse appear to indicate that mouse NM does not bioactivate p-cresidine. This could be examined by assessment of DNA adduct formation. p-Cresidine is only weakly active or negative in most genotoxicity assays (Ashby et al., 1991), but did produce DNA damage in mouse bladder mucosa measured by the single cell gel electrophoresis (Comet) assay (Sasaki et al., 1998). No epidemiologic report was available from the IARC review of p-cresidine (International Agency for Research on Cancer, 1982b) and none was found in a literature search. Nevertheless, p-cresidine was listed as reasonably anticipated to be a human carcinogen by NTP (National Toxicology Program, 2005). 2,6-Dimethylaniline (2,6-DMA, Figure 17), or 2,6-xylidine, is used mainly in dye manufacture. It is also present in tobacco smoke (Bryant et al., 1988). It is a biotransformant of lidocaine (Figure 18; (Keenaghan and Boyes, 1972). 2,6-DMA induced nasal tumors in rats (National Toxicology Program, 1990b) in a complex study in which 5-week-old CD rats were given 2,6-DMA in the diet at 0, 300, 1000, or 3000 ppm and at 16 weeks they were mated and the females continued on the diets during pregnancy and lactation. The offspring, after weaning at 3 weeks, were then continued on the same diets as their parents for 104 weeks. Nasal adenomas and carcinomas were seen in both male (1000 and 3000 ppm) and female (3000 ppm only) progeny, along with some other tumors. No findings were reported on the parental generation. No carcinogenicity study in mice has been reported. Since the compound is volatile and loss from the feed occurred, there was discussion in the report (National Toxicology Program, 1990b) that the neoplasms may have been a result of inhalation exposure. This issue has not been resolved, but subsequent findings support the likelihood of the nasal effects being due to systemic distribution. For example, 2,6-DMA was reported to have ‘promoting’ activity in the OM when given in the diet at 3000 ppm for 52 weeks after N-bis(2-hydroxypropyl)nitrosamine (Koujitani et al., 1999). Rather than representing promoting activity, however, this effect may be due to enhancement of carcinogenicity by toxicity or a syncarcinogenic effect (Williams and Iatropoulos, 2001) resulting from summation of the genotoxicity of the 2,6-DMA together with that of the nitrosamine, as reported for other combinations of DNA-reactive carcinogens (Williams and Furuya, 1984).
The nasal carcinogenic activity of 2,6-DMA is highly structure-dependent, in as much as the 2,4- and 2,5-DMAs, although carcinogenic in rats and mice, were not reported to produce nasal neoplasms (Weisburger et al., 1978). Studies on the genotoxicity of 2,6-DMA, reviewed by IARC (International Agency for Research on Cancer, 1993a), showed it to be weakly mutagenic in vitro but not in vivo. Nevertheless, covalent binding to DNA of 2,6-DMA in rat NM has been demonstrated using radiolabeled compound (Short et al., 1989) and by nucleotide postlabeling (Gonçalves et al., 2001; Jeffrey et al., 2002). 2,6-DMA is likely bioactivated through N-hydroxylation (Figure 17), as with other aromatic amines (Miller, 1998). Synthetically prepared N-hydroxy-2,6-DMA was highly mutagenic to S. typhimurium and bound to DNA in vitro without bioactivation (Gonçalves et al., 2001; Jeffrey et al., 2002). 2,6-DMA is a biotransformant of lidocaine (Figure 18), a widely used local anesthetic, which is also used to treat arrhythmias, and is used as a veterinary drug. Following the administration of lidocaine, 2,6-DMA has been identified in human tissues (Keenaghan and Boyes, 1972; Parker et al., 1996), and urine (Nelson et al., 1977). Also, hemoglobin adducts have been identified by GC/MS in humans receiving lidocaine (Bryant et al., 1994). 2,6-DMA has also been found in milk from cows and humans treated with lidocaine (Puente and Josephy, 2001). Following iv administration of lidocaine to patients, methemoglobinemia has been observed (Weiss et al., 1987), which is consistent with the formation of an N-hydroxy biotransformant (Kiese, 1966). No study on the carcinogenicity of lidocaine is described in the summary of preclinical studies in the PDR (2006), although biotransformants, presumably 2,6-DMA, are described as carcinogenic in laboratory animals (PDR, 2006). No report of an association of nasal tumors in humans has been made for exposure to either 2,6-DMA or lidocaine. This is in spite of theoretical concerns raised by the fact that lidocaine is used as a nasal spray for the treatment of migraine headaches, as well as a local anesthetic during nasal surgery (Genter, 2004). Smokers exhibit NM alterations and have increased risk of sinonasal squamous cell carcinoma (Feron et al., 2001). In cigarette smokers, in addition to the well established increases in 4-aminobiphenyl-hemoglobin adducts, 2,6-DMA-hemoglobin adducts have been reported to be increased (Bryant et al., 1988), although, curiously, adducts were 3 times higher in nonsmokers. 2,6-Diethylaniline and 2,4,6-trimethylaniline (mesidine), are structurally similar to 2,6-DMA There are few studies on 2,6-diethylaniline despite the fact that it is a key biotransformant of the chloroacetanilide herbicides (Feng et al., 1990), discussed next, which are carcinogenic to the nasal cavity. 2,4,6-trimethylaniline produced mouse liver DNA strand breaks in the single cell gel electrophoresis assay (Przybojewska, 1999), but was not mutagenic and evidence for its carcinogenic activity was considered not evaluable by IARC (International Agency for Research on Cancer, 1982a). A structural analog, 2,4,5-trimethylaniline, has limited evidence for carcinogenicity in rodents, and was weakly mutagenic to Salmonella with bioactivation (Kugler-Steigmeier et al., 1989). In contrast to the weak mutagenicity in bacteria, 2,4,5-trimethylaniline was quite mutagenic in the Drosophila wing spot assay and to cultured fibroblasts (Kugler-Steigmeier et al., 1989). o-Anisidine (2-methoxyaniline), which lacks the 5-methyl group of p-cresidine, was carcinogenic in rodents and positive for bacterial mutagenicity (International Agency for Research on Cancer, 1982c), although negative for DNA adduct formation (Ashby et al., 1994). p-Anisidine (4-methoxy aniline) was not adequately tested in rats and was negative for bacterial mutagenicity (International Agency for Research on Cancer, 1982c). Aniline, the simplest aromatic amine, and a variety of its derivatives are in use in industry. It had carcinogenic activity in rats, producing mainly splenic sarcomas, whereas it was not carcinogenic in mice (International Agency for Research on Cancer, 1987a). This probably reflects the greater susceptibility of rats to aromatic amine-induced methemoglobinemia (Kiese, 1966), which leads to splenic congestion and necrosis due to the culling of abnormal erythrocytes. In in vitro genotoxicity assays, aniline was negative (Williams et al., 1989; Przybojewska, 1999). Several related monocyclic aromatic amines have also been tested. p-Chloroaniline has shown genotoxic activity (Williams et al., 1989) and was carcinogenic in both rats and mice (International Agency for Research on Cancer, 1993b), although not in the nasal cavity (Chhabra et al., 1991). Thirty-seven aniline derivatives have been tested in the hepatocyte DNA repair assay (Yoshimi et al., 1988), which responds specifically to DNA-reactive chemicals (Williams et al., 1989). Of these, 6 were positive, as follows: 2,4-DMA, 2,4,6-trimethylaniline (mesidine), 3,5-diaminobenzoic acid, 3,4-diaminochlorobenzene, 2-chloro-4-methylaniline and 4-chloro-N-methylaniline. Of these 6, some produced liver or bladder tumors, but not nasal tumors. Most notably, aniline and o-chloro-, o-methoxy-, o-ethyl-derivatives were all negative, as was phenetidine and 2,4,6-trichloroaniline. Given the high degree of correlation between positive results in hepatocyte DNA repair and carcinogenicity (Williams et al., 1989), it is expected that the positive chemicals would have carcinogenic activity at some site. Study of NM DNA adducts could provide insight as to whether this tissue would be a target for any of these chemicals. o-Toluidine (2-methylaniline), as its hydrochloride, induced tumors at several sites, but not in the nasal cavity, in both male and female rats and mice starting at doses of up to 6,000 ppm (Weisburger et al., 1978; National Toxicology Program, 1979a; Hecht et al., 1982). IARC (International Agency for Research on Cancer, 1987d) concluded that there was sufficient evidence for carcinogenicity in animals. More recent additional studies in mice, rats and dogs confirmed the carcinogenicity of o-toluidine, but did not report on the occurrence of nasal neoplasms (Pliss, 2004). In genotoxicity assays, it produced a variety of positive effects, but results were often conflicting (International Agency for Research on Cancer, 1987d). o-Toluidine is a biotransformant of the local anesthetic prilocaine (PDR, 2006). In humans, hemoglobin adducts of o-toluidine have been reported in smokers and at lower levels in non-smokers (Bryant et al., 1988), and in workers in a chemical manufacturing facility (Ward et al., 1996). It is possible that the substitutions in the anilines that lacked activity in the NM do not permit facile enzymic N-hydroxylation or sulfation to take place, as occurs with 2,6-DMA. In addition, if such biotransformants are formed, it may be that they require sufficient intrinsic reactivity to bind to DNA in order to be carcinogenic. As with 2,6-DMA, no report of any nasal effect in humans was found for these other anilines. The structurally more complex polycyclic aromatic amines, for example the carcinogens β-naphthylamine, 4-aminobiphenyl, or 4,4'-methylene-bis(2-chloroaniline) (MOCA), are well-documented genotoxins (McQueen and Williams, 1990; Kadlubar and Badawi, 1995), as are the related complex heterocyclic derivatives formed as food pyrolysis products (Sugimura et al., 2000), as a consequence of N-hydroxylation and bioactivation (Miller, 1998). The simpler monocyclic derivatives discussed here generally show less genotoxicity. Based on a literature search, no polycyclic aromatic amine has been reported to produce nasal tumors in rodents, although in chronic toxicity studies a deliberate search for neoplasia may not always have been made. However, polycyclic aromatic amines can be bioactivated in the NM, since 4,4'-methylene-bis(2-chloroaniline) was found to bind to rat NM DNA (Jeffrey et al., 2002). Benzene is the simplest aromatic hydrocarbon (Figure 19). It is widely used as an industrial solvent and is present in gasoline (~1%), automobile emissions, cigarette smoke, drinking water and a variety of foods. For most people, the level of exposure to benzene is likely to be higher from inhalation than ingestion. Benzene has been tested extensively for carcinogenicity and while positive, only 1 study reported nasal tumors resulting from oral administration (Maltoni et al., 1989). Benzene at a dose of 500 mg/kg bw for 78 weeks to both SD and Wistar rats was reported to induce nasal tumors, as well as Zymbal gland carcinomas, carcinomas of the oral cavity, skin, forestomach, and mammary glands, angiosarcomas of the liver, hemolymphoreticular neoplasias, tumors of the lung, and possibly hepatomas (Maltoni et al., 1989). Another study, however, using doses up to 200 mg/kg bw for 2 years, did not report nasal tumors (Huff et al., 1989). Homogenates from the NM had greater biotransformation capacity towards benzene than those from the liver (Low et al., 2003). Moreover, high concentrations of benzene were found to produce an inflamatory response and DNA fragmentation in cultured human nasal respiratory mucosa (Gosepath et al., 2003). However, unlike the related compounds naphthalene and bromobenzene discussed here, no report of rodent nasal cytotoxicity by benzene was found. Benzene yielded mixed results in genotoxicity test systems (International Agency for Research on Cancer, 2000b), with the exception of bone marrow. DNA binding was either absent or extremely low in several tissues in rats (Reddy et al., 1989), although binding was reported under extreme conditions (twice-daily treatment for 1 to 7 days with 440 mg/kg benzene) (Bodell et al., 1996). A scheme for the biotransformation of benzene based upon (Snyder and Hedli, 1996) is shown in Figure 19. The benzene biotransformant hydroquinone also has been tested for carcinogenicity in mice and rats with some evidence for increased kidney neoplasms in rats and liver neoplasms in mice at high doses, but without effect in the NM (National Toxicology Program, 1989). Epigenetic modes of action for the hydroquinone tumor increases have been proposed (Whysner et al., 1995). Most human studies have not reported nasal effects of benzene (International Agency for Research on Cancer, 1987b). A cohort study of Chinese workers in occupations where benzene exposure is possible reported a suggestive increase in nasopharyngeal cancer mortality in males, but not females (Yin et al., 1996), and without a positive trend for cumulative exposure (Hayes et al., 1996). Moreover, nasopharyngeal cancer is endemic in China (see later) and confounding factors may be involved. The authors noted that tobacco use is frequent among Chinese men, but not women. Tobacco smoke contains benzene, 2,6-DMA, and carcinogenic nitrosamines (see later).
Chloroacetanilides
Chloroacetanilides were negative in most genotoxicity assays, including the comet assay for DNA damage in NM (Heydens et al., 1999). A study of binding of radiolabeled alachlor to rat nasal tissues showed minimal binding to DNA, although binding to proteins was substantial and twice that found in the liver (Heydens et al., 1999). At carcinogenic dosages, alachlor induced a substantial increase in cell proliferation in rat OE, but not RE at 60 days of dosing (Heydens et al., 1999). The oncogenicity of chloroacetanilides has been linked to the formation of 2,6-dialkylbenzoquinoneimines (Feng et al., 1990; Li et al., 1992) (Figure 20), which can b |
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Abstract
Introduction
-tocopherol (
