Differential effects of dopamine melanin on norharman-induced toxicity in PC12 cells.

The food contaminant norharman structurally resembles MPTP a compound that selectively damages pigmented brain areas. Both compounds are sequestered and retained in melanin-containing neurons. The aim of the study was to examine whether intracellular melanin can modulate the toxicity of norharman in melanin-loaded PC12 cells. Dopamine melanin protected against norharman-induced upregulation of grp78, activation of caspase 3 and necrosis at low concentrations (5 and 50 microM). In contrast, at a high conentration (500 microM) there was a significantly increased expression of grp78, hsp90 and caspase 3 and a disassociation of melanin aggregates leading to dispersal of granules to swollen neurite terminals. In human populations, a long-term low-level exposure to toxicants with a high affinity to melanin will probably result in accumulation in melanin-containing neurons in vivo. Our data suggest that accumulation of a neurotoxicant in melanin-loaded cells may lead to increased cell stress, apoptotic signaling and disassociation of melanin aggregates.

Norharman-induced motoric impairment in mice: neurodegeneration and glial activation in substantia nigra.

The beta-carboline norharman is present in cooked food and tobacco smoke and show structural resemblance to the neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. C57BL/6 mice were injected subcutaneously with norharman (3 and 10 mg/kg) twice per day for five consecutive days. Eighteen hours after the last dose an increased expression of glial fibrillary acidic protein and fluoro-jade staining were demonstrated whereas the number of tyrosine hydroxylase positive cells were unchanged in the substantia nigra. Two weeks after the last treatment a decreased motor activity was observed whereas cognitive functions remained intact. In cultured PC12 cells norharman treatment induced mitochondrial dysfunction and increased the number of caspase-3 and TUNEL-positive cells. The results demonstrate that norharman induced apoptosis in cultured cells as well as early neurodegeneration, glial activation and sustained motor deficits in mice and suggest that exposure to norharman may contribute to idiopathic Parkinson's disease.

Long-term retention of neurotoxic beta-carbolines in brain neuromelanin.

beta-Carbolines show structural resemblance to the neurotoxic N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and are metabolized to mitochondrial toxicants. Humans are continuously exposed to low levels of beta-carbolines through cooked food, coffee, alcoholic beverages and tobacco smoke. beta-Carbolines have previously been detected in higher levels in the pigmented substantia nigra than in the cortex of humans. The distribution of 3H-labelled harman and norharman in the brain of pigmented and albino mice and in frogs (a species having neuromelanin) was studied by tape-section and light-microscopic autoradiography. Furthermore, the binding of these beta-carbolines to dopamine-melanin and melanin granules from Sepia officinalis was examined. The results revealed a high affinity binding to melanin and a long-term retention (up to 30 days) in pigmented tissues, including neuromelanin-containing neurons of frogs after a single injection. The role of long-term exposure to food-related beta-carbolines and a retention of these compounds in pigment-containing neurons in the induction of idiopathic Parkinson's disease should be further considered.

Effects of lactic acid bacteria on the uptake and distribution of the food mutagen Trp-P-2 in mice.

BACKGROUND: Lactic acid bacteria have been reported to have antimutagenic and anticarcinogenic properties in vivo and in vitro. Lactobacillus acidophilus and Bifidobacterium longum have earlier been shown to bind the food mutagen Trp-P-2 in vitro. METHODS: The influence of oral supplementation with L. acidophilus NCFB 1748 and B. longum BB 536 on the uptake and distribution of 14C-labelled Trp-P-2 in several mouse tissues was quantified by liquid scintillation measurements and examined by tape section autoradiography (gives an unbiased qualitative registration of differences in overall tissue distribution) in the present investigation. Furthermore, the effect of 13-naphthoflavone (BNF), a cytochrome P4501A (CYP1A)-inducing agent, on the distribution of 14C-labelled Trp-P-2 was examined. RESULTS: After oral (6 mg/kg; 5 microCi) or iv (1.2 mg/kg; 1 microCi) administration of 14C-labelled Trp-P-2, high levels of radioactivity were observed in the bile, urine and contents of the gastrointestinal tract. Lower levels were present in the liver, lung, kidney, intestines, brown fat, submaxillary salivary gland and thymus. In mice supplemented with lactic acid bacteria there was a significantly decreased level (29%-73%) of radioactivity in the lung, thymus, liver, kidney, submaxillary salivary gland and small intestine as compared with controls. In mice pretreated with BNF, a low but distinct localization of radioactivity in the lung was observed, whereas no similar localization occurred in controls. CONCLUSIONS: The results suggest that (i) there is a decreased bioavailability of the Trp-P-2 in the majority of the tissues examined in bacteria supplemented mice and (ii) there is a low but distinct CYP1A-dependent activation of Trp-P-2 in the lung of BNF-treated mice.

Drug targeting to the brain: transfer of picolinic acid along the olfactory pathways.

Picolinic acid (PA) protects against quinolinic acid- and kainic acid-induced neurotoxicity in the brain. To study the uptake of PA to the brain, we administered [3H]PA via a unilateral nasal instillation or iv injection to mice. Autoradiography demonstrated a rapid uptake of radioactivity in the olfactory nerve layer and in the ipsilateral olfactory bulb (OB) following nasal instillation of [3H]PA. After 4 h, there was a high level of radioactivity in the central parts of the ipsilateral OB and olfactory peduncle. Moreover, iv injection of [3H]PA demonstrated a selective uptake and retention of radioactivity in the OB. Gas chromatography-mass spectrometry (GC-MS) demonstrated the presence of PA and PA-glycine conjugate in the OB. In mice with reduced peripheral olfactory innervations there was a decreased uptake of [3H]PA in the OB as compared to controls suggesting that an intact olfactory neuroepithelium is a prerequisite for an uptake of PA to the OB. There is an increased interest in brain targeting of drugs with limited ability to pass the blood-brain barrier. The present results demonstrate that PA fulfils structural requirements for a transfer along the olfactory pathways to the brain.

Differential response of cultured human umbilical vein and artery endothelial cells to Ah receptor agonist treatment: CYP-dependent activation of food and environmental mutagens.

In the present study, 7-ethoxyresorufin O-deethylase (EROD), 7, 12-dimethylbenz[a]anthracene (DMBA)-hydroxylase, and covalent binding of (3)H-labeled 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole ((3)H-Trp-P-1) and (3)H-DMBA were examined in human umbilical vein endothelial cells (HUVEC) and human umbilical artery endothelial cells (HUAEC) exposed to the aryl hydrocarbon (Ah) receptor agonist beta-naphthoflavone (BNF) or vehicle only. The results revealed a marked induction of enzymatic activity in BNF-treated HUVEC compared with vehicle-treated cells, whereas no similar response was observed in BNF-treated HUAEC. EROD, DMBA hydroxylase, and covalent binding of (3)H-Trp-P-1 and (3)H-DMBA in BNF-treated HUVEC were reduced in the presence of the CYP1A inhibitor ellipticine. Addition of other CYP1A inhibitors alpha-naphthoflavone, miconazole, 1-ethynylpyrene, 1-(1-propynyl)pyrene), or the CYP1A substrate ethoxyresorufin to the incubation buffer of BNF-treated HUVEC reduced covalent binding of (3)H-Trp-P-1 by 93-98%. Western blot analysis confirmed an induction of CYP1A1 in BNF-treated HUVEC, but not in BNF-treated HUAEC. CYP1A1 was, however, detected in both vehicle- and BNF-treated HUAEC. The results showed that BNF exposure induced CYP1A1 and metabolic activation of xenobiotics in HUVEC, whereas the catalytic activity remained low in BNF-treated HUAEC. Our results suggest that endothelial lining of human veins may be a target for adverse effects of xenobiotics activated into reactive metabolites by Ah receptor-regulated enzymes. Several studies have detected CYP1A1 in endothelial linings, whereas expression of CYP1A2 and CYP1B1 seems to be negligible at this site. This suggests that the metabolic activation and covalent binding of (3)H-Trp-P-1 and (3)H-DMBA in HUVEC are most likely mediated by CYP1A1.

CYP1A-dependent activation of xenobiotics in endothelial linings of the chorioallantoic membrane (CAM) in birds.

Metabolic activation of the heterocyclic amine 3-amino -1,4-dimethyl-5 H-pyrido[4,3-b]indole (Trp-P-1) and 7-ethoxyresorufin O-deethylase (EROD) activity were examined in the chorioallantoic membrane (CAM) of 15-day-old chicken and 18-day-old eider duck embryos. The embryos were pretreated with an Ah receptor agonist, i.e. beta-naphthoflavone (BNF) or 3,3',4,4',5-pentachlorobiphenyl (PCB 126), or vehicle in ovo. BNF and PCB 126 induced EROD activity and covalent binding of [3H]Trp-P-1 seven- to tenfold in the CAM of chicken embryos. In the CAM of eider duck embryos, which are known to be nonresponsive to coplanar PCBs, PCB 126 treatment had no effect on EROD activity or covalent binding of [3H]Trp-P-1 whereas BNF treatment increased these activities five- and threefold, respectively. Light microscopic autoradiography was used to identify the cellular localization of covalent binding of [3H]Trp-P-1 in the CAM. Preferential binding was observed in endothelial cells in intraepithelial capillaries in the chorionic epithelium and in blood vessels in the mesenchymal layer. The addition of the CYP1A inhibitor ellipticine abolished the covalent binding of [3H]Trp-P-1 in the CAM of BNF- and PCB 126-treated chicken and eider duck embryos. The results suggest that CYP1A-dependent metabolic activity can be induced in blood vessel endothelia in the CAM of bird embryos following exposure to Ah receptor agonists and that the CAM may be a target tissue for CYP1A-activated environmental pollutants. Furthermore, the highly vascularized CAM could be used as a model for studies of Ah receptor-mediated alterations in the vasculature.

Persistent olfactory mucosal metaplasia and increased olfactory bulb glial fibrillary acidic protein levels following a single dose of methylsulfonyl-dichlorobenzene in mice: comparison of the 2,5- and 2, 6-dichlorinated isomers.

Histopathology was used to characterize long-term toxic effects in the olfactory system following a single ip dose (4-65 mg/kg) of methylsulfonyl-2,6-dichlorobenzene, (2,6-(diCl-MeSO(2)-B)), in female NMRI mice. The effects of 2,6-(diCl-MeSO(2)-B) and its 2, 5-chlorinated isomer, (2,5-(diCl-MeSO(2)-B)), on the levels of glial fibrillary acidic protein (GFAP; a biomarker for neurotoxicity) in different brain regions were examined by an enzyme-linked immunosorbent assay (ELISA). The histopathologic effects of 2, 6-(diCl-MeSO(2)-B) were dose-, time-, and tissue-dependent. At the highest doses (16-65 mg/kg), the initial effect of 2, 6-(diCl-MeSO(2)-B) was necrosis of the Bowman's glands, followed by a sequence of secondary events including degeneration of the olfactory neuroepithelium, repopulation of the basement membrane by a ciliated respiratorylike epithelium, fibrosis and ossification in the lamina propria, formation of bilateral polyps, angiogenesis, and disappearance of nerve bundles. Remodeling was most pronounced in the dorsal meatus of the olfactory mucosa and persisted for the duration of the experiment (46 weeks). A dose-dependent induction of GFAP in the olfactory bulb of mice treated with 2,6-(diCl-MeSO(2)-B) was observed at all doses examined (16-65 mg/kg). GFAP levels were highest 2 weeks after treatment (eightfold induction at 65 mg/kg) and then gradually decreased to normal within 26 weeks. The 2, 5-substituted isomer (65 mg/kg) did not induce GFAP in the olfactory bulb and or toxicity in the olfactory mucosa. In conclusion, a single dose of 2,6-(diCl-MeSO(2)-B) results in persistent metaplasia and remodeling of the olfactory mucosa, and a long-lasting but transient induction of GFAP in the olfactory bulb. It is proposed that methylsulfonyl-2,6-dichlorobenzene may serve as an experimental tool with a unique ability to produce persistent primary and/or secondary lesions in the olfactory system of mice.

Binding of the potent allergen hexahydrophthalic anhydride in the mucosa of the upper respiratory and alimentary tract following single inhalation exposures in guinea pigs and rats.

Hexahydrophthalic anhydride (HHPA; CAS No. 13149-00-3) is a highly allergenic compound commonly used in the chemical industry. Guinea pigs and rats were exposed to [3H2]HHPA by inhalation for 3-8 h and were killed at various intervals during 7 days. The tissue distribution of non-volatile and covalently bound radioactivity was studied by autoradiography. Tissue bound radioactivity was mainly found in the mucosa of the upper respiratory airways, whereas negligible levels were observed in the lungs. In addition, tissue bound radioactivity was present in the gastrointestinal tract and conjunctiva. Moreover, in the cortex of the kidneys in rats, but not in guinea pigs, a low level of tissue bound radioactivity was found. The radioactivity in the tissues persisted for at least 7 days after the end of exposure. Plasma proteins and soluble proteins from trachea, lung, and kidney from [3H2]HHPA-exposed animals were separated by gel filtration. The radioactivity in dialysed plasma was mainly found in the same fractions as albumin. The soluble proteins from trachea, lung, and kidney in both rats and guinea pigs showed a similar pattern as found in blood. The radioactivity in dialysed plasma from both guinea pigs and rats seemed to decay according to a two-compartment model. The non-extractable binding of [3H2]HHPA in the upper respiratory airways and conjunctiva may be of relevance for symptoms in workers with allergy, since they mainly develop symptoms and signs from the nose and eyes.

Localization and comparative toxicity of methylsulfonyl-2,5- and 2,6-dichlorobenzene in the olfactory mucosa of mice.

Several methylsulfonyl (MeSO2) metabolites formed from chlorinated aromatic hydrocarbons have been identified in human milk, lung, and body fat, as well as in the tissues of Baltic grey seals and arctic polar bears. The tissue localization and nasal toxicity of two methylsulfonyl-substituted dichlorobenzenes (diCl-MeSO2-B), with the chlorine atoms in the 2,5-, and 2,6- positions, were investigated in female NMRI and C57B1 mice. Using tape-section autoradiography, animals dosed i.v. with 14C-labeled 2,5-, or 2,6-(diCl-MeSO2-B) showed a preferential uptake of radioactivity in the olfactory mucosa and the tracheobronchial epithelium. Histopathology showed that 2,6-(diCl-MeSO2-B) is a potent toxicant that induces necrosis in the olfactory mucosa following a single dose as low as 4 mg/kg (i.p. injection), whereas 2,5-(diCl-MeSO2-B) induced no signs of toxicity in the olfactory mucosa at doses as high as 130 mg/kg (i.p. injection). Necrosis of the Bowman's glands was the first sign of 2,6-(diCl-MeSO2-B)-induced toxicity followed by degeneration of the neuroepithelium, which implies that the Bowman's gland may be the primary site of toxicity and degeneration of the neuroepithelium may be a secondary effect. Administration of the parent compounds, 1,3-dichlorobenzene and 1,4-dichlorobenzene, or the chlorinated analog 1,2,3-trichlorobenzene (85, 85, and 105 mg/kg, respectively; i.p. injection), induced no signs of toxicity in the olfactory mucosa. These and previous results suggest that 2,6-positioned chlorine atoms and an electron withdrawing substituent in the primary position is an arrangement that predisposes for toxicity in the olfactory mucosa.

Methimazole toxicity in rodents: covalent binding in the olfactory mucosa and detection of glial fibrillary acidic protein in the olfactory bulb.

Methimazole is an antithyroid drug reported to affect the sense of smell and taste in humans. The aim of the present study was to examine the distribution and effects of methimazole on the olfactory system in rodents. Autoradiography showed a selective covalent binding of 3H-labeled methimazole in the Bowman's glands in the olfactory mucosa, bronchial epithelium in the lungs, and centrilobular parts of the liver following an iv injection in mice. Histopathology showed an extensive lesion in the olfactory mucosa that was efficiently repaired 3 months after two consecutive ip doses of methimazole. The effect of methimazole on various brain regions was studied by determining levels and location of glial fibrillary acidic protein (GFAP). The results showed a threefold increase of GFAP in the olfactory bulb 2 weeks after treatment with methimazole whereas no change was observed 4 days after treatment. Pretreatment of mice with thyroxine did not protect against the methimazole-induced toxicity in the olfactory mucosa and bulb. In contrast, pretreatment with the cytochrome P450 inhibitor metyrapone completely prevented the covalent binding and toxicity of methimazole in the olfactory mucosa and bulb. The present results suggest that the methimazole-induced toxicity in the olfactory mucosa is mediated by a cytochrome P450-dependent metabolic activation of the compound into reactive metabolites that are bound to various tissues including the olfactory mucosa. The increase of GFAP in the olfactory bulb of methimazole-treated mice is suggested to be a secondary phenomenon due to the primary damage in the olfactory mucosa.

Transfer of some carboxylic acids in the olfactory system following intranasal administration.

The uptake of [14C]benzoic acid, 4-chloro[14C]benzoic acid, [3H]phthalic acid and [14C]salicylic acid in the nasal passages and brain was determined following a unilateral intranasal instillation in mice. An uptake of radioactivity from the nasal mucosa to the ipsilateral olfactory bulb was observed up to 4 h after administration following intranasal instillation of these carboxylic acids whereas the level was low in the contralateral olfactory bulb. Autoradiography of mice given [14C]benzoic acid and [14C]salicylic acid by intranasal instillation showed a preferential localization of radioactivity in the axonal and glomerular layer of the olfactory bulb 1 h after the administration. Four hours after administration the radioactivity was present as a gradient from the axonal layer towards the center of the olfactory bulb. Pretreatment of mice with a compound known to damage the olfactory neuroepithelium resulted in a decreased uptake of [14C]benzoic acid in the olfactory bulb. Thin layer chromatography of supernatants from the ipsilateral olfactory bulbs of mice given [14C]benzoic acid by nasal instillation indicated that the radioactivity in the bulbs represented unchanged compound. These results suggest that there is a transfer of some aromatic carboxylic acids in the olfactory pathways.

Basal and induced EROD activity in the chorioallantoic membrane during chicken embryo development.

The chorioallantoic membrane (CAM) is a highly vascularized tissue that takes part in the respiratory exchange of gases through the eggshell. Although the CAM may be exposed to environmental contaminants, its response to pollutants has not been studied. We examined the cytochrome P4501A (CYP1A)-catalyzed deethylation of 7-ethoxyresorufin (EROD) in the CAM during chicken embryo development. EROD was constitutively present and was inducible by the aryl hydrocarbon (Ah) receptor agonist 3,3',4,4',5-pentachlorobiphenyl (PCB 126). Our results suggest the CAM as a first line of defence of the avian embryo against toxic compounds, but also as a target for CYP1A-activated chemicals.

Localization of cytochrome P4501A1 and covalent binding of a mutagenic heterocyclic amine in blood vessel endothelia of rodents.

Immunohistochemistry was used to examine the cellular localization of cytochrome P4501A1 (CYP1A1) in various types of endothelial linings in muscle tissues of rats and mice treated with the Ah receptor agonist beta-naphthoflavone (BNF). In addition, light microscopic autoradiography was used to localize sites of metabolic activation of 3H-labeled Trp-P-1 (3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole), a heterocyclic amine known to be metabolized by CYP1A1, in rodent tissue slices. The results showed a colocalization of CYP1A1 immunoreactivity and covalent binding of 3H-Trp-P-1 in endothelial linings of capillaries and veins of heart, skeletal muscle, and uterus in BNF-treated rodents, indicating the presence of catalytically active CYP1A1 at these sites. The immunohistochemical staining and covalent binding of 3H-Trp-P-1 in endothelia of arteries and arterioles was generally weak with the exception of uterine arterioles. In lymph nodes of BNF-treated rats, there was an intense CYP1A1 staining of high endothelial venules. The results suggest that endothelial linings of capillaries and veins in muscle tissues but also uterine arterioles and high endothelial venules in lymph nodes may be targets for CYP1A1-mediated metabolic products of endogenous and exogenous substances following exposure to CYP1A1 inducing agents.

Induction of ethoxyresorufin O-deethylase (EROD) and endothelial activation of the heterocyclic amine Trp-P-1 in bird embryo hearts.

The xenobiotic-metabolizing activity of avian heart was investigated in chicken and Eider duck embryos exposed to aryl hydrocarbon (Ah) receptor agonists in ovo. Both beta-naphthoflavone (BNF) and 3,3',4,4',5-pentachlorobiphenyl (PCB 126) induced 7-ethoxyresorufin O-deethylase (EROD) activities in chicken embryo hearts whereas Eider duck embryos only responded to BNF. The differential responses of chicken and Eider duck embryos were used to examine the involvement of Ah receptor-mediated enzyme induction in the activation of the environmental and food mutagen 3-amino- 1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1). As determined by light microscopic autoradiography, there was a highly selective binding of non-extractable 3H-Trp-P-1-derived radioactivity in endothelial cells of large vessels and capillaries in hearts of BNF- and PCB 126-treated chicken embryos. No binding occurred at these sites in vehicle-treated controls. There was also a strong endothelial binding of 3H-Trp-P-1 in hearts of BNF-treated Eider duck embryos whereas no binding occurred in hearts of PCB 126-treated Eider duck embryos. A positive correlation between induction of EROD activity and covalent binding of 3H-Trp-P-1 to protein in heart homogenates from BNF- and PCB 126-treated chicken and Eider duck embryos was also observed. The results suggest a cytochrome P450 1A (CYP1A)-mediated activation of Trp-P-1 in avian heart endothelial cells although involvement of other Ah receptor-regulated enzymes is also possible. We propose that heart endothelial cells may be targets for bioactivation and toxicity of environmental contaminants in birds exposed to Ah receptor agonists.

Metabolism-dependent activation and toxicity of chemicals in nasal glands.

The mucosae of the nasal passages contain a large amount of glands which express secretory proteins as well as phase I and phase II biotransformation enzymes. In this review the metabolic activation, covalent binding and toxicity of chemicals in the Bowman's glands in the olfactory mucosa, in the sero-mucous glands in the nasal septum and in the lateral nasal glands and maxillary glands around the maxillary sinuses are discussed. Light microscopic autoradiographic studies have demonstrated a selective covalent binding of nasal toxicants and carcinogens such as halogenated hydrocarbons and N-nitrosamines, especially in the Bowman's glands following a single systemic exposure, suggesting a high rate of metabolic activation of chemicals in these glands. Special attention is put on the herbicide dichlobenil which induces necrosis in the olfactory mucosa following a cytochrome-P450-mediated metabolic activation and covalent binding in the Bowman's glands.

Effects of dichlobenil on ultrastructural morphology and cell replication in the mouse olfactory mucosa.

The herbicide dichlobenil (2,6-dichlorobenzonitrile) is a tissue-specific inducing necrosis in the olfactory mucosa. Transmission electron microscopy showed vacuolations (1 hr) and necrosis (4 hr) in the Bowman's glands following intraperitoneal injection of dichlobenil (25 mg/kg) in C57B1 mice, whereas no lesions were noted in the olfactory neuroepithelium at these timepoints. Twenty-four hr following injection (25 mg/kg), most of the propria and the olfactory epithelium were severely damaged; the basal lamina, nerves, and blood vessels in the lamina propria, however, remained intact. Following a lower dose (12 mg/kg), the lesions were not as pronounced. The effects of dichlobenil (6, 12, and 25 mg/kg) on cell replication in the olfactory mucosa, as determined by incorporation of 3H-thymidine 3 days later, were more pronounced in the lamina propria than in the neuroepithelium and occurred at a lower dose in the lamina propria than in the neuroepithelium. Together these studies support the previous proposal that dichlobenil induces a primary damage in the Bowman's glands.

Cell- and tissue-specific metabolic activation of chemicals as determined by autoradiography: in vitro-in vivo correlations.

Numerous chemicals require metabolic activation to exert toxicity. Such metabolism-dependent toxicity may be due to the formation of electrophiles that bind irreversibly to cellular macromolecules in the vicinity of their site of formation. A high cellular concentration of a covalently bound metabolite may thus imply a target site for toxicity. Autoradiography at different levels of resolution is useful to determine cell-specific covalent metabolite binding of radiolabelled chemicals, both in vivo and in vitro. Using this approach, cell-specific toxicants such as dichlobenil (olfactory mucosa) and MeSO(2)-DDE (adrenal cortex) have been identified. In addition, unforeseen sites of local metabolism of toxic chemicals have been demonstrated. In our experience, the in vitro-in vivo correlations are often good, while in some cases results obtained in vivo cannot be reproduced in vitro. The inability of tissue slices to account for in vivo events may depend on metabolism in non-target tissues and differences in tissue disposition in vivo and in vitro. Examples are given to demonstrate the utility of autoradiography in vitro and in vivo to trace metabolism-dependent binding and toxicity in airway epithelia, vascular endothelia and adrenal zona fasciculata cells.

3-Aminobenzamide: effects on cytochrome P450-dependent metabolism of chemicals and on the toxicity of dichlobenil in the olfactory mucosa.

Treatment with 3-aminobenzamide, known as an inhibitor of poly(ADP-ribose)polymerease, decreased the toxicity and covalent binding of the herbicide dichlobenil (2,6-dichlorobenzonitrile; 12 mg/kg; i.p.) in the mouse olfactory mucosa. In vitro studies showed that 3-aminobenzamide markedly reduced the NADPH-dependent covalent binding of [14C]dichlobenil and the hydroxylation of p-nitrophenol which have previously been suggested to be mediated by a common form of cytochrome P450 (P450) in rat olfactory microsomes (Eriksson and Brittebo, Chem.-Biol. Interact. 94,183-196, 1995). Furthermore, 3-aminobenzamide markedly reduced the P450-dependent metabolic activation of [3H]NNK (4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone) in rat olfactory microsomes and slightly decreased the P450 2B1-dependent pentoxyresorufindealkylase activity in liver microsomes of phenobarbital-treated rats. The present results suggest that 3-aminobenzamide is also an inhibitor of P450 and that the lack of toxicity of dichlobenil in the olfactory mucosa of 3-aminobenzamide-treated mice is related to a decreased metabolic activation of dichlobenil at this site. Further experiments showed that there was no evidence for a binding of [14C]dichlobenil metabolites to calf thymus DNA or a formation of mutagenic dichlobenil metabolites in Ames' Salmonella assay when dichlobenil was incubated in the presence of homogenates of the olfactory mucosa. Finally, analysis of proteins from olfactory microsomes incubated with [14C]dichlobenil using SDS-PAGE/fluorography revealed a binding of metabolites to all major proteins. Addition of glutathione or the P450-inhibitor metyrapone prevented the binding, suggesting the formation of relatively stable electrophilic products which can leave the activating enzyme and then unselectively bind to the major olfactory microsomal proteins.

Effects of the herbicide chlorthiamid on the olfactory mucosa.

Chlorthiamid (2,6-dichlorothiobenzamide) and its major metabolite 2,6-dichlorobenzonitrile are olfactory toxicants with a high in vivo covalent binding in the olfactory mucosa of mice. This study showed that the cytochrome P450 (P450) inhibitors, metyrapone and sodium-diethyldithiocarbamate, abolished the chlorthiamid-induced toxicity (12 mg/kg; 0.06 mmol/kg) in C57B1/6 mice suggesting a P450-dependent toxicity. Incubation of [14C]-labelled chlorthiamid with rat olfactory microsomes showed a low NADPH-dependent oxidative covalent binding which was only 3-fold higher than that in liver microsomes. Thus the results do not support a major in situ metabolic activation of chlorthiamid and it is suggested that metabolic activation of the major chlorthiamid metabolite (2,6-dichlorobenzonitrile) is responsible for most of the covalent binding and toxicity of chlorthiamid at this site in vivo. Thiobenzamide (16 mg/kg; 0.12 mmol/kg), a dechlorinated chlorthiamid-analog, induced no marked morphological changes in the olfactory mucosa demonstrating that chlorines in the 2,6-position are important for the chlorthiamid-induced toxicity at this site.