CYP2F2-generated metabolites, not styrene oxide, are a key event mediating the mode of action of styrene-induced mouse lung tumors

Styrene induces lung tumors in mice but not in rats. Although metabolism of styrene to 7,8-styrene oxide (SO) by CYP2E1 has been suggested as a mediator of styrene toxicity, lung toxicity is not attenuated in CYP2E1 knockout mice. However, styrene and/or SO metabolism by mouse lung Clara cell-localized CYP2F2 to ring-oxidized cytotoxic metabolite(s) has been postulated as a key metabolic gateway responsible for both lung toxicity and possible tumorigenicity. To test this hypothesis, the lung toxicity of styrene and SO was evaluated in C57BL/6 (WT) and CYP2F2(−/−) knockout mice treated with styrene (400 mg/kg/day, gavage, or 200 or 400 mg/kg/day, ip) or S- or R-SO (200 mg/kg/day, ip) for 5 days. Styrene treated WT mice displayed significant necrosis and exfoliation of Clara cells, and cumulative BrdU-labeling index of S-phase cells was markedly increased in terminal bronchioles of WT mice exposed to styrene or S- or RSO. In contrast, Clara and terminal bronchiole cell toxicity was not observed in CYP2F2(−/−) mice exposed to either styrene or SO. This study clearly demonstrates that the mouse lung toxicity of both styrene and SO is critically dependent on metabolism by CYP2F2. Importantly, the human isoform of CYP2F, CYP2F1, is expressed at much lower levels and likely does not catalyze significant styrene metabolism, supporting the hypothesis that styrene-induced mouse lung tumors may not quantitatively, or possibly qualitatively, predict lung tumor potential in humans.     

Metabolism of styrene by mouse and rat isolated lung cells.

Styrene is pneumotoxic in mice. It is metabolized by pulmonary microsomes of both mouse and rat to styrene oxide (SO), presumed to be the toxic metabolite of styrene, and known to be genotoxic. To determine which pulmonary cell types are responsible for styrene metabolism, and which cytochromes P450 are associated with the bioactivation of styrene, we isolated enriched fractions of mouse and rat Clara and type II cells in order to determine the rate of styrene metabolism, with and without chemical inhibitors. Mouse Clara cells readily metabolized styrene to SO. Diethyldithiocarbamate, a CYP2E1 inhibitor, caused less inhibition of SO formation in Clara cells isolated from mice than previously found with pulmonary microsomes. As in microsomes, 5-phenyl-1-pentyne, a CYP2F2 inhibitor, inhibited the formation of both enantiomers. alpha-Naphthoflavone, a CYP1A inhibitor, did not inhibit SO formation in Clara cells. alpha-Methylbenzylaminobenzotriazole, a CYP2B inhibitor, exhibited minimal inhibition of SO production at 10 microM and less at 1 microM. The microsomal and isolated cell studies indicate that CYP2E1 and CYP2F2 are the primary cytochromes P450 involved in pulmonary styrene metabolism. Styrene metabolizing activity was much greater in Clara cells than in type II pneumocytes, which demonstrated essentially no activity. Styrene-metabolizing activity was several-fold higher in the mouse than in rat Clara cells. The more pneumotoxic and genotoxic form, R-SO, was preferentially formed in mice, and S-SO was preferentially formed in rats. These findings indicate the importance of Clara cells in styrene metabolism and suggest that differences in metabolism may be responsible for the greater susceptibility of the mouse to styrene-induced toxicity.     

Ring-oxidized metabolites of styrene contribute to styrene-induced Clara-cell toxicity in mice

Styrene produced cytotoxicity in the terminal bronchioles of mice, but not rats, due to metabolites produced in situ by CYP2F2 metabolism. It has generally been presumed that styrene toxicity is mediated by styrene 7,8-oxide, but styrene oxide is not much more toxic than styrene. In contrast, ring-oxidized metabolites (4-vinylphenol or its metabolites) induce much greater toxicity. Administration of 4-vinylphenol results in pneumotoxicity, based on analysis of bronchoalveolar lavage fluid (BALF) at a 5- to 10 fold lower dose than does styrene oxide. In the current research, studies demonstrated that ip administration of 4-vinylphenol for 14 consecutive days at dosages of 6, 20, or 60 mg/kg/d (split into 3 doses) produced cytotoxicity in the terminal bronchioles of mice, but not rats. While higher doses of 4-vinylphenol produced adverse effects in both liver and lung, no liver toxicity was seen in mice exposed to 60 mg/kg/d for 14 d. Approximately 4 d was required for BALF parameters to return to normal following a single administration of 4-vinylphenol. These studies add further support for the role of ring-oxidized metabolites in the pneumotoxicity induced by styrene in mice and the lack thereof in rats.     

Mouse specific lung tumors from CYP2F2-mediated cytotoxic metabolism: An endpoint/toxic response where data from multiple chemicals converge to support a mode of action

It is proposed that metabolism of several structurally-related chemicals by CYP2F isoforms of the cytochromes P450 family results in a cytotoxicity-driven mode of action in organs high in CYP2F; namely, CYP2F2 in nasal and lung tissue in mice and CYP2F4 in nasal tissues in rats. Importantly, the CYP2F1 isozyme expressed in humans appears to have a low capacity to metabolize these compounds. In mice, the resultant cytotoxicity and subsequent regenerative hyperplasia is hypothesized drive an increase in lung tumors that are mostly benign and are not life shortening. Although a complete picture of the mode of action has not been developed in any one model compound, data from the individual compounds can be combined to synthesize and reinforce confidence in the CYP2F toxicity hypothesis. For coumarin, naphthalene, and styrene, inhibition of toxicity with inhibition of CYP2F2 has been demonstrated. Rat CYP2F4 appears to be equally active in metabolizing these chemicals; however, CYP2F4 occurs to a much lower extent in rat Clara cells and levels of metabolites produced are not sufficient to cause lung cytotoxicity. Human lungs contain far fewer of Clara cells than rats or mice, and human lung microsomes failed to, or only marginally, metabolize these compounds. In addition, the human lung differs markedly from the mouse lung in the morphology of its Clara cells, which make humans much less sensitive than mice to toxicity due to reactive metabolites. The absence of a role for CYP2E1-generated metabolites (primarily alkyl oxidation vs. ring-oxidation) in mouse pulmonary effects was demonstrated by the lack of protection from styrene toxicity by CYP2E1 inhibitor, or reduction of toxicity in CYP2E1-knockout mice, and lack of lung toxicity of the primary metabolite of ethylbenzene. The chemicals used as examples of this mode of action generally are negative in standard genotoxicity assays. Apart from increased SCE, no consistent pattern in genotoxicity results was found among these chemicals. Thus, while lung tumors from bronchiolar cell cytotoxicity are theoretically possible in humans, it is unlikely that metabolism by CYP2F1 would produce levels of cytotoxic metabolites in human lungs sufficient to result in lung cytotoxic responses and thus tumors. Therefore, it is unlikely several chemicals that cause mouse lung tumors via CYP2F2 metabolism will cause lung tumors in humans.     

Comparison of styrene and its metabolites styrene oxide and 4-vinylphenol on cytotoxicity and glutathione depletion in Clara cells of mice and rats

Styrene is a widely used compound in the manufacturing industry. In mice and rats, it is both hepatotoxic and pneumotoxic. It causes lung tumors in mice, but not in rats. The Clara cell is the main target for the toxicity of styrene and its metabolites, and it also has the greatest activity for styrene metabolism. Therefore, Clara cells isolated from CD-1 mice and Sprague-Dawley rats were used to compare the cytotoxicities induced by styrene and its metabolites. The cytotoxicity of styrene was greater in vitro than that of its metabolites styrene oxide (racemic, R- and S-) and 4-vinylphenol in contrast with what has been observed in vivo in previous studies on hepatotoxicity and pneumotoxicity. Susceptibility of rats to styrene and its metabolites are 4-fold less than that observed with mice. Glutathione levels were also measured in mice following addition of the chemicals in vitro and treatment of the CD-1 mice in vivo. Decreases in glutathione concentrations were seen even at doses which did not cause the death of mouse Clara cells. Significant decreases in glutathione were observed 3h after treatment with racemic SO and R-SO. At 12h, rebound effects were seen for all compounds, with all but R-SO rebounding above controls. These studies suggest that in vitro cytotoxicity of styrene and its metabolites does not strictly follow in vivo effects and that decreases in mouse glutathione levels may be related to oxidative stress.     

The role of cytochromes P-450 in styrene induced pulmonary toxicity and carcinogenicity.

Exposure of CD-1 mice to atmospheres of 40 and 160 ppm styrene, daily for up to 10 days, caused pulmonary toxicity characterised by focal loss of cytoplasm and focal crowding of non-ciliated Clara cells, particularly in the terminal bronchiolar region. The toxicity was accompanied by an increase in cell replication rates in terminal and large bronchioles of mice exposed for 3 days or longer. The toxicity and increased cell replication were no longer apparent after a 2-day break in exposure, but re-occurred when exposure was resumed. Similar effects were seen in mice given oral doses of 10, 100 or 200 mg/kg styrene, daily for 5 days. Increases in cell replication rates were seen in the terminal bronchioles in mice dosed with 100 and 200 mg/kg styrene, but not 10 mg/kg. Toxicity was limited to 3 to 10 animals in the 200 mg/kg group. Neither morphological nor cell proliferation effects were seen in the alveolar region of the mouse lung in any of these studies, nor were any effects observed in the lungs of CD rats exposed to 500 ppm styrene for up to 10 days. The pulmonary toxicity and increased cell division seen in mice, but not rats, correlates with the known species differences in pulmonary carcinogenicity of styrene, suggesting that the acute and chronic responses are causally related. 5-Phenyl-1-pentyne was shown to inhibit the pulmonary cytochrome P-450 metabolism of styrene in vivo. Cell replication rates in the lungs of mice treated with this inhibitor and exposed to styrene were comparable with controls demonstrating that the pulmonary effects of styrene on the mouse lung are caused by a metabolite of styrene, probably styrene oxide. The risks associated with exposure to styrene appear to correlate well with the metabolic capacity of the lung.     

Comparison of styrene oxide enantiomers for hepatotoxic and pneumotoxic effects in microsomal epoxide hydrolase-deficient mice

Styrene is hepatotoxic and pneumotoxic in mice. Styrene oxide, the active metabolite, is detoxified via hydrolysis by microsomal epoxide hydrolase (mEH). Racemic styrene oxide was previously found to be more lethal and produced increased toxicity in mEH-/- mice compared to wild-type mice. The hepatotoxicity and pneumotoxicity of the R- and S-styrene oxide (SO) enantiomers were compared in wild-type and mEH-deficient mice (mEH-/-). Twenty-four hours following administration of 150 mg/kg ip, neither enantiomer produced hepatotoxicity, but S-SO was more pneumotoxic. However, in mEH-/- mice R-SO produced greater decreases in hepatic glutathione levels 3 h after administration. The basis for the unusual greater toxicity of S-SO, rather than the generally more toxic R-SO, in mEH-/- mice may be related to differences in detoxification by EH.     

Four-Day Repeated Inhalation and Recovery Study of Methyl lsocyanate in F344 Rats and B6C3F1 Mice

F344 rats and B6C3F1 mice of both sexes were exposed by inhalationto 0, l,and 3 ppm methyl isocyanate (MIC) for 4 consecutivedays (6 hr/day) followed by a recovery period of 91 days. Fivemice and rats/sex/group except the 3 ppm group (5 rats/ sexon Day 7 and 2 males on Day 28) were killed on Days 7, 28, 49,and 91 after the exposure and examined histopathologically.Forty-nine of 56 male rats, 51 of 56 female rats, and 1 of 56male mice in the 3 ppm group died by 28 days; early death animalswere also examined histologically. Exposure-related changesoccurred in rats and mice of both sexes in the 3 ppm group only.Lesions of the nasal cavity in rats and mice were characterizedby regeneration of the olfactory and respiratory epithelia secondaryto epithelial erosion. By Day 28 the olfactory and respiratoryepithelia in mice appeared normal, while in rats incompleteregeneration of the olfactory epithelium was still present.Regeneration of the respiratory epithelium in the trachea ofrats occurred in the 3 ppm group and the epithelium appearedto return to normal by Day 28. Lung lesions in rats consistedof mural and/or intraluminal fibrosis secondary to extensiveerosion of the respiratory epithelium in the major bronchi tothe terminal bronchioles. Acute inflammation of the small airways,occasional hyaline membranes of alveolar walls, and pulmonaryatelectasis were also seen. Alveolar fibrosis was observed inrats found dead from Day 14 on and in male rats killed on Day28. Atrophy of the thymus and spleen, atrial thrombosis of theheart, and hepatocellular necrosis were frequently seen in ratsdying following MIC exposure. The lung lesions in mice werequalitatively similar to those in rats, but were restrictedto the major bronchi. Minimal intraluminal or mural fibrosiswas still present in mice on Day 91. In a separate study, asingle 6-hr exposure of five male rats to 3 ppm MIC was followedby a recovery period of 7 days. The lesions of the respiratorysystem were essentially the same as those in the 3 ppm groupkilled on Day 7 after the 4-day repeated exposure of MIC, butthe alveolar lesions were more severe.     

Biochemical quantitation and histochemical localization of carboxylesterase in the nasal passages of the Fischer-344 rat and B6C3F1 mouse

Inhalation exposure of rats and mice to glycol ether acetates and acrylate esters causes degeneration of the olfactory epithelium but not of the respiratory epithelium. Since these compounds are metabolized via carboxylesterase to acids that are toxic to the olfactory epithelium, the activity and cellular distribution of carboxylesterase in the nasal passages of rats and mice were studied. Olfactory mucosal carboxylesterase in both rats and mice was found to have a Vmax value for the hydrolysis of p-nitrophenyl butyrate approximately 3 to 6 times larger than that for respiratory mucosa. Similarly, the second-order rate constant for binding and catalysis, V/K, was approximately four times greater in olfactory mucosa than in respiratory mucosa of both rats and mice. These data demonstrate that the olfactory mucosa of rats and mice hydrolyze carboxylesters more efficiently than the respiratory mucosae. Enzyme histochemistry was employed to identify the individual cells within the respiratory and olfactory mucosae which contain carboxylesterase activity. All cell types of the respiratory epithelium had some carboxylesterase activity, with varying intensities between individual cell populations. Ciliated and cuboidal epithelial cells were most active in this region. In the olfactory mucosa, however, Bowman's glands stained most intensely, sustentacular cells demonstrated moderate activity, and no activity was detectable in olfactory sensory cells. Together, these data quantitate carboxylesterase activity in nasal mucosal homogenates and localize the enzyme in individual cell types. The data suggest that olfactory mucosa may metabolize carboxylesters to acids more readily than respiratory mucosa. However, such metabolism does not occur in the target cell population, the olfactory sensory neurons, raising the possibility of intercellular migration of toxic acid metabolites.     

Localization of oestradiol in the rat nasal mucosa

The metabolism of 4-14C-oestradiol in the rodent respiratory tract has been investigated in vitro. Using thin-layer radiochromatography, at least 3 and 2 metabolites were found at incubation with slices from the rat olfactory mucosa and lung, respectively. Autoradiography of the nasal region of rats injected intravenously with 4-14C-oestradiol showed a high level of radioactivity in the mucosa covering the respiratory region, and in the subepithelial parts of the olfactory mucosa. Autoradiography of rat and mouse lung slices incubated with 4-14C-oestradiol showed that a selective localization of radioactivity occurred in the bronchial mucosa. The radioactivity in the mucosa of the respiratory tract was reversibly bound and could be extracted with organic solvents.     

Histochemical localization of aldehyde dehydrogenase in the respiratory tract of the Fischer-344 rat

Acetaldehyde, a nasal carcinogen and respiratory tract irritant, is metabolized by aldehyde dehydrogenase. The localization of aldehyde dehydrogenase in individual cells of the nasal passages, trachea, and lungs of the Fischer-344 rat was determined histochemically using a cold glycol methacrylate embedding procedure. Aldehyde dehydrogenase activity was detected principally in the nasal respiratory epithelium, especially in the supranuclear cytoplasm of ciliated epithelial cells while olfactory epithelium was almost devoid of enzyme activity. Epithelial cells of the trachea demonstrated little detectable aldehyde dehydrogenase activity, however, the Clara cells of the lower bronchioles showed remarkably high activity. These results corroborate previous biochemical findings and extend them by histochemically identifying particular aldehyde dehydrogenase-positive cell types within the nasal respiratory epithelium. The distribution of nasal lesions induced by acetaldehyde correlated with regional aldehyde dehydrogenase deficiencies suggesting that regional susceptibility to the toxic effects of acetaldehyde may be due, at least in part, to a lack of aldehyde dehydrogenase in the susceptible regions. Furthermore, aldehyde dehydrogenase activity in the Clara cells indicates a possible site for metabolism of aldehydes which penetrate to the lower airways.     

Effects of styrene and its metabolites on different lung compartments of the mouse--cell proliferation and histomorphology

Styrene is not carcinogenic in rats but has caused pneumotoxicity and increased lung tumors after inhalation in mice. This study investigated whether styrene-7,8-oxide, ring-oxidized, and side-chain hydroxylated styrene metabolites induce cell proliferation, apoptosis, pathological changes, and glutathione depletion in mice lungs. Intraperitoneal treatment with phenylacetaldehyde and phenylacetic acid (3 x 100 mg/kg b.w./day) increased the levels of apoptosis and cell proliferation in the alveoli without producing any effects in the terminal bronchioli, the target site of tumor formation in mice. Only styrene-oxide (SO) at 3 x 100 mg/kg b.w./day and 4-vinyl-phenol (4-VP) at 3 x 35 and 3 x 20 mg/kg b.w./day, respectively, caused up to 19-fold increases in cell proliferation in the large/medium bronchi and terminal bronchioles; marginal increases in alveolar cell proliferation were noted with SO (1.6-fold) but not with 4-VP. These compounds also caused glutathione depletion in the bronchiolar epithelium and histomorphological changes of the bronchiolar epithelium in large and medium bronchi and terminal bronchioles. Changes were characterized by flattened cells and a loss of the typical bulging of the "dome-shaped" Clara cells, suggesting that Clara cells were primary target cells. The specific reactions of mouse lung to SO and 4-VP could serve as a verifiable hypothesis for the different response of rats and mice with regard to tumor formation.     

Chronic toxicity/oncogenicity study of styrene in CD-1 mice by inhalation exposure for 104 weeks

Groups of 70 male and 70 female Charles River CD-1 mice were exposed whole body to styrene vapor at 0, 20, 40, 80 or 160 ppm 6 h per day 5 days per week for 98 weeks (females) or 104 weeks (males). The mice were observed daily; body weights, food and water consumption were measured periodically, a battery of hematological and clinical pathology examinations were conducted at weeks 13, 26, 52, 78 and 98 (females)/104 (males). Ten mice of each gender per group were pre-selected for necropsy after 52 and 78 weeks of exposure and the survivors of the remaining 50 of each gender per group were necropsied after 98 or 104 weeks. An extensive set of organs from the control and high-exposure mice were examined histopathologically, whereas target organs, gross lesions and all masses were examined in all other groups. Styrene had no effect on survival in males. Two high-dose females died (acute liver toxicity) during the first 2 weeks; the remaining exposed females had a slightly higher survival than control mice. Levels of styrene and styrene oxide (SO) in the blood at the end of a 6 h exposure during week 74 were proportional to exposure concentration, except that at 20 ppm the SO level was below the limit of detection. There were no changes of toxicological significance in hematology, clinical chemistry, urinalysis or organ weights. Mice exposed to 80 or 160 ppm gained slightly less weight than the controls. Styrene-related non-neoplastic histopathological changes were found only in the nasal passages and lungs. In the nasal passages of males and females at all exposure concentrations, the changes included respiratory metaplasia of the olfactory epithelium with changes in the underlying Bowman's gland; the severity increased with styrene concentration and duration of exposure. Loss of olfactory nerve fibers was seen in mice exposed to 40, 80 or 160 ppm. In the lungs, there was decreased eosinophilia of Clara cells in the terminal bronchioles and bronchiolar epithelial hyperplasia extending into alveolar ducts. Increased tumor incidence occurred only in the lung. The incidence of bronchioloalveolar adenomas was significantly increased in males exposed to 40, 80 or 160 ppm and in females exposed to 20, 40 and 160 ppm. The increase was seen only after 24 months. In females exposed to 160 ppm, the incidence of bronchiolo-alveolar carcinomas after 24 months was significantly greater than in the controls. No difference in lung tumors between control and styrene-exposed mice was seen in the intensity or degree of immunostaining, the location of tumors relative to bronchioles or histological type (papillary, solid or mixed). It appears that styrene induces an increase in the number of lung tumors seen spontaneously in CD-1 mice.     

Respiratory pathology in rats and mice after inhalation of 1,2-dibromo-3-chloropropane or 1,2 dibromoethane for 13 weeks

Seventy F344 rats and 144 B6C3F1 mice were subdivided into seven groups. Three groups were each exposed via inhalation to 1, 5, or 25 ppm of 1,2-dibromo-3-chloropropane (DBCP) for 6 h per day, 5 days per week for 13 weeks. Three additional groups were each similarly exposed to 3, 15, or 75 ppm of 1,2-Dibromoethane (EDB). The remaining group was exposed to room air under the same conditions. At 13 weeks, rats and mice showed severe necrosis and atrophy of the olfactory epithelium in the nasal cavity after inhalation of 5 or 25 ppm DBCP and 75 ppm EDB. Lower concentrations induced squamous cell metaplasia, hyperplasia and cytomegaly of the epithelium of the respiratory nasal turbinals. Squamous metaplasia, hyperplasia and cytomegaly of the epithelium was also seen in larynx, trachea, bronchi and bronchioles. Other compound related toxic lesions in rats were seen in the liver, kidney and testes.     

Immunoblot analysis and immunohistochemical characterization of CYP2A expression in human olfactory mucosa

The aim of the present study was to further characterize the expression of the CYP2A genes in human nasal mucosa. Fetal nasal tissues at 12-26 weeks of gestational age and surgical biopsy tissues from various regions of nasal cavity of adult patients were studied to determine whether CYP2A proteins can be detected by immunoblot in adults, whether higher levels of CYP2A proteins are present in adult than in fetal nasal mucosal microsomes, and whether CYP2A13 mRNA is more abundant than CYP2A6 mRNA in fetal nasal mucosa. In adults, immunoblot analysis detected CYP2A proteins in microsomes of the olfactory region from 8 of 10 individuals, but in none of the nasal microsomes of the respiratory region from 47 patients. Quantitative immunoblot analysis confirmed that CYP2A proteins are selectively expressed in the olfactory region in both adult and fetal tissues. Interestingly, the levels of CYP2A proteins in nasal microsomes were generally higher in fetuses than in adults. In the fetus, the level of CYP2A13 mRNA was much higher than that of CYP2A6 mRNA, as has been previously found in adult nasal mucosa. Immunohistochemical studies confirmed that, in the fetus, the CYP2A proteins are expressed in the supporting cells in the olfactory epithelium and in the Bowman’s glands in the lamina propria. The prenatal expression of the CYP2A proteins in the olfactory mucosa suggests potential risks of developmental toxicity from maternally derived xenobiotics, since both CYP2A6 and CYP2A13 are known to be efficient in the metabolic activation of tobacco-specific nitrosamines and other respiratory toxicants.     

Respective roles of CYP2A5 and CYP2F2 in the bioactivation of 3-methylindole in mouse olfactory mucosa and lung: studies using Cyp2a5-null and Cyp2f2-null mouse models

The aim of this study was to determine whether mouse CYP2A5 and CYP2F2 play critical roles in the bioactivation of 3-methylindole (3MI), a tissue-selective toxicant, in the target tissues, the nasal olfactory mucosa (OM) and lung. Five metabolites of 3MI were identified in NADPH- and GSH-fortified microsomal reactions, including 3-glutathionyl-S-methylindole (GS-A1), 3-methyl-2-glutathionyl-S-indole (GS-A2), 3-hydroxy-3-methyleneindolenine (HMI), indole-3-carbinol (I-3-C), and 3-methyloxindole (MOI). The metabolite profiles and enzyme kinetics of the reactions were compared between OM and lung, and among wild-type, Cyp2a5-null, and Cyp2f2-null mice. In lung reactions, GS-A1, GS-A2, and HMI were detected as major products, and I-3-C and MOI, as minor metabolites. In OM reactions, all five metabolites were detected in ample amounts. The loss of CYP2F2 affected formation of all 3MI metabolites in the lung and formation of HMI, GS-A1, and GS-A2 in the OM. In contrast, loss of CYP2A5 did not affect formation of 3MI metabolites in the lung but caused substantial decreases in I-3-C and MOI formation in the OM. Thus, whereas CYP2F2 plays a critical role in the 3MI metabolism in the lung, both CYP2A5 and CYP2F2 play important roles in 3MI metabolism in the OM. Furthermore, the fate of the reactive metabolites produced by the two enzymes through common dehydrogenation and epoxidation pathways seemed to differ with CYP2A5 supporting direct conversion to stable metabolites and CYP2F2 supporting further formation of reactive iminium ions. These results provide the basis for understanding the respective roles of CYP2A5 and CYP2F2 in 3MI's toxicity in the respiratory tract.     

Modification of the metabolism and toxicity of styrene and styrene oxide in hepatic cytochrome P450 reductase deficient mice and CYP2F2 deficient mice

Styrene causes toxicity in both the lung and the liver. The study of the relationship of this toxicity to the metabolism of styrene has been aided by the use of knockout mice for both bioactivation and detoxification pathways. It has been hypothesized that CYP2E1 is primarily responsible for styrene bioactivation in mouse liver and CYP2F2 in mouse lung. Two knockout strains were used in the current studies. Mice deficient in hepatic cytochrome P450 reductase had much less hepatic metabolism of styrene to styrene oxide. Styrene (600 mg/kg, i.p.) caused significant hepatotoxicity, as determined by serum sorbitol dehydrogenase and glutathione levels, in the wild-type but not in the knockout mice. It caused lung toxicity, as determined by protein levels, cell number, and lactate dehydrogenase activity in the bronchioalveolar lavage fluid of wild-type mice, but this effect was less in the knockout mice. In CYP2F2 knockout mice there was only a small decrease in the hepatic metabolism of styrene but a very large decrease in pulmonary metabolism. As expected the CYP2F2 knockout and wild-type mice were equally susceptible to styrene-induced hepatotoxicity, but the knockout mice were less susceptible to styrene-induced pneumotoxicity. Although the results are inconsistent with the simple hypothesis that styrene pneumotoxicity is due to the bioactivation of styrene to styrene oxide by CYYP2F2, they demonstrate the importance of both liver and lung in the metabolism of styrene, but additional pharmacokinetic studies are needed to help clarify the relationship between target organ metabolism and susceptibility.     

Methyl methacrylate toxicity in rat nasal epithelium: studies of the mechanism of action and comparisons between species

Female F344 rats exposed to 200 ppm methyl methacrylate for 6 h developed a lesion in the nasal olfactory epithelium which was characterised by degeneration and atrophy. The severity of the lesion was markedly reduced by pre-treatment of the rats with an intraperitoneal dose of 100 mg/kg bis-(p-nitrophenyl)phosphate, an inhibitor of carboxylesterase enzymes, thus demonstrating that the lesion is caused by the carboxylesterase mediated metabolism of methyl methacrylate to methacrylic acid, an irritant and corrosive metabolite. The distribution of the carboxylesterases in nasal tissues has been investigated and the metabolism of methyl methacrylate to methacrylic acid has been compared in rat, hamster and human nasal tissue fractions in vitro. Histocytochemistry showed that the carboxylesterases are heavily localised in the sustentacular cells and Bowman's glands of the rat olfactory region, but are more generally distributed in human olfactory epithelium. Consistent with this, the enzyme activity in all three species was higher in fractions prepared from olfactory tissue than from respiratory tissue, 3-fold in rat and human and 12-fold in the hamster. The maximum rates (V(max)) of metabolism in rat and hamster olfactory tissue fractions were comparable, whereas those in human olfactory tissue fractions were at least 13-fold lower. The rate of metabolism in rat olfactory tissue was also comparable to that in rat liver whereas in humans, the rate in olfactory tissue was 500-fold lower than that in the liver. In respiratory tissues, the rate in humans was at least 6-fold lower than that in the rat. These results suggest that humans are significantly less sensitive than rodents to the nasal toxicity of methyl methacrylate.