Celia J. Reed

Xenobiotic Metabolizing Enzymes of the Kidney

The kidney possesses most of the common xenobiotic metabolizing enzymes, and is thus able to make an important contribution to the bodys metabolism of drugs and foreign compounds. An overview of the renal localization, catalytic activity, developmental regulation, induction, and sex and species differences for the key enzymes involved in phase I and phase II of xenobiotic metabolism is presented. In general, the catalytic activities of the various renal enzymes are lower than those of the liver, although there are exceptions, such as the enzymes involved in the processing of glutathione conjugates to their mercapturic acids. Xenobiotic metabolizing enzymes are not evenly distributed along the nephron; cytochromes P-450 and those enzymes involved in the conjugation of glutathione, glucuronic acid, or sulfate are primarily localized in the proximal tubules. However, some isozymes of cytochrome(s) P-450 and glutathione S-transferases are selectively localized in cells of the thick ascending limb and distal tubules, whereas prostaglandin H synthase is concentrated in the collecting ducts in the medulla. Thus, the proximal tubule, the principal site of xenobiotic biotransformation, is particularly susceptible to chemical insult, and the localization of prostaglandin synthase in the inner medulla and papilla may be a contributary factor to the toxicity produced by chemicals in this part of the nephron. Many of the enzymes discussed, in addition to metabolizing foreign compounds, have important endogenous functions in the kidney, such as the regulation of salt and water balance and the synthesis of vitamin D.

Immunohistochemical localisation of six glutathione S-transferases within the nasal cavity of the rat

Many xenobiotics induce lesions within the nasal cavity of experimental animals which are site specific. This site selectivity may be due to regional deposition within the nasal cavity and/or the localisation of biotransformation enzymes. We have developed methodology which allows immunohistochemical localisation of xenobiotic biotransformation enzymes in transverse sections of the rat nasal cavity identical to those normally taken for pathological examination. We report the application of this methodology to six isoenzymes of the glutathione S-transferases (GSTs). All six isoenzymes were predominantly located within olfactory epithelium covering the ethmoturbinates (levels III and IV) and extending forwards into the dorsal meatus (level II). Squamous and transitional epithelia showed little or no staining while respiratory epithelium was weakly stained. Within the respiratory epithelium only the ciliated columnar cells and, to a lesser extent, some of the seromucous glands contained GSTs. Within olfactory epithelium the sustentacular cells, basal cells and subepithelial glands all stained positive for GSTs. The different cell types of olfactory epithelium preferentially express different GST isoenzymes: 1-1 and 2-2 were predominantly located in the subepithelial glands; 3-3, 4-4 and 8-8 in sustentacular and basal cells; 7-7 in basal cells.

Antioxidant status of the rat nasal cavity

Despite extensive interest in the rodent nasal cavity as a target organ for toxicity, there is very limited information regarding nasal defenses against oxidative stress and xenobiotic-derived oxidants. Using immunohistochemistry, we have examined the distribution of Cu,Zn and Mn superoxide dismutase (SOD), catalase, glutathione (GSH) peroxidase, and DT-diaphorase in rat nasal tissues. In addition, we have determined the concentrations of ascorbate and alpha-tocopherol and the activities of SOD (combined Cu,Zn and Mn forms), catalase, GSH peroxidase, GSH reductase, and DT-diaphorase in nasal respiratory epithelium (RE), olfactory epithelium (OE), and in lung. Immunohistochemistry demonstrated that all four enzymes were similarly distributed, with the greatest staining intensity in dorsal-medial regions of the nasal cavity. In respiratory epithelium, ciliated columnar cells and subepithelial glands stained positively, while in olfactory tissue the enzymes were detected in the sustentacular cells and Bowmans glands. With the exception of SOD, enzyme activities were higher in RE than OE, while concentrations of ascorbate and alpha-tocopherol were higher in OE than RE. With the exception of catalase, nasal activities were either higher than or comparable to those of the lung. Thus, the rat nasal cavity appears to be well protected against oxidative damage.

Olfactory toxicity of methyl iodide in the rat

The monohalomethanes (methyl iodide, methyl bromide and methyl chloride) are widely used industrial methylating agents with pronounced acute and chronic toxicity in both experimental animals and man. Recently inhalation exposure of rats to methyl bromide has been shown to result in severe olfactory toxicity. This study examined the effects on the rat nasal cavity of inhalation of methyl iodide (100 ppm for 0.5–6 h), and demonstrated that methyl iodide is a more potent olfactory toxin than methyl bromide. Within the nasal cavity the olfactory epithelium was the principle target tissue, and it was only at high doses (600 ppm.h) that limited damage to transitional epithelium occurred. The squamous and respiratory epithelia were consistently unaffected. Within olfactory epithelium the sustentacular cells were the primary cellular target and damage to sensory cells appeared to be a secondary event. Methyl iodide induced olfactory damage was reversible, and 2 weeks after exposure almost complete repair had taken place.

The role of glutathione S-transferase- and cytochrome P450-dependent metabolism in the olfactory toxicity of methyl iodide in the rat

Abstract The aim of this study was to investigate the role of metabolic activation in the olfactory toxicity of methyl iodide (MeI). Adult male rats were exposed via nose-only inhalation to 100ppm MeI for 0–6h, and non-protein sulphydryl (NP-SH) concentrations determined in selected tissues. Depletion of NP-SH occurred in all tissues, but was most marked and rapid in the respiratory epithelium of the nasal cavity and the kidney. Olfactory, lung and liver NP-SH levels were affected to a lesser extent, and those of the brain declined by only 20–30% over the whole time course. In order to modulate glutathione (GSH) status, animals were pre-treated with (1) phorone plus l-buthionine sulphoximine (BSO), which depleted NP-SH levels in all the tissues examined, or (2) the isopropyl ester of GSH (IP-GSH), which was shown to replenish NP-SH concentrations in all tissues except the liver of animals previously administered phorone. When animals were pre-treated with phorone plus BSO and then exposed to 100ppm MeI for 2h, there was a potentiation of the toxicity of MeI as judged by the clinical observations on the animals. In contrast, treatment with IP-GSH prior to and during exposure to MeI for 4h afforded a marked protection to the olfactory epithelium. In order to inhibit cytochromes P450, animals were pre-treated with cobalt protoporphyrin IX. This decreased hepatic cytochrome P450 concentrations by >90%, but when animals were then exposed to 100ppm MeI for 4h there was no effect on the severity of the olfactory lesion. These results indicate that conjugation of MeI with GSH is a detoxification rather than an activation pathway. Also, there is no major role for cytochrome P450-dependent oxidation in the development of the olfactory lesion.

Investigations of the pathways of toxicity of methyl iodide in the rat nasal cavity

The monohalomethane methyl iodide (MeI) is a site specific toxin within the nasal cavity of the rat, selectively damaging the olfactory epithelium (OE) whilst respiratory epithelium (RE) is spared. The aim of this study was to investigate the rates and routes of metabolism of MeI within the nasal cavity, in order to understand the reasons for the observed site-selectivity. Cytosolic glutathione S-transferases (GSTs) of both OE and RE catalysed the conjugation of MeI with glutathione (GSH), but rates were 4-fold higher in OE than RE. The product of this reaction was confirmed as S-methyl GSH. In both OE and liver the GST catalysing the conjugation of MeI was shown to belong to the theta class. No cytochrome P450-dependent oxidation of MeI to formaldehyde could be detected in incubations containing hepatic or olfactory microsomes. Intact nasal turbinates were incubated with [14C]-MeI, and a dose- and time-dependent covalent binding of MeI to olfactory protein was demonstrated. The rates of protein methylation were found to be similar in OE and RE. Thus the only parameter that correlates with the site-selectivity of the observed lesion is the rate of conjugation of MeI with GSH. Whether toxicity is due to production of a reactive metabolite or GSH depletion per se, remains to be elucidated.

Methyl iodide toxicity in rat cerebellar granule cells in vitro: the role of glutathione

The monohalomethane methyl iodide (MeI) is toxic to a number of organ systems including the central nervous system. Clinical symptoms of neurotoxicity suggest that the cerebellum is the target within the brain, and we have now modelled the toxicity of MeI in cultured rat cerebellar granule cells. Cytotoxicity is maximal 24 h after a 5 min exposure to MeI, and the EC50 for MeI under these conditions was calculated to be 1.6 mM. The glutathione S-transferase (GST) dependent metabolism of MeI was investigated in these cultures. There was a marked decrease in intracellular glutathione (GSH) 15 min after exposure to MeI, and GSH concentrations then increased, reaching 130% of control levels 7 h after exposure. To investigate the role of conjugation with GSH in the toxicity of MeI, GSH levels were modulated prior to exposure. Depletion of GSH exacerbated the cytotoxicity of MeI while provision of a bioavailable source of GSH was protective. Inclusion of antioxidants [vitamin E, butylated hydroxytoluene (BHT) or desferrioxamine mesylate (DF)] also protected against the cytotoxicity of MeI. Our in vitro data suggest that MeI is conjugated with GSH in the cerebellum, and the resulting extensive depletion of GSH may be the first step en route to toxicity, rendering the tissue susceptible to methylation and/or oxidative stress.

A rat nasal epithelial model for predicting upper respiratory tract toxicity: in vivo–in vitro correlations

An in vitro model of the rat nasal cavity has been used to compare the responses of nasal tissues in vitro, using loss of intracellular ATP and potassium as indices of toxicity, with the pathological changes occurring following in vivo exposure to four test compounds. Turbinates were incubated in vitro with the test compounds for 4 h, for 24 h or for 4 h followed by 20 h in fresh medium. Titanium dioxide caused little or no loss of ATP in either olfactory epithelium (OE) or respiratory epithelium (RE). Sodium carbonate decreased olfactory, but not respiratory ATP, while acetic acid and 3-methylindole markedly decreased ATP in both tissues. Intracellular potassium concentrations were generally affected to a lesser degree. In vivo, no morphological changes were observed in the nasal cavity following inhalation exposure to either titanium dioxide or sodium carbonate. Inhalation of acetic acid resulted in a very focal lesion in the RE of the dorsal meatus of level 1, while administration of 3-methylindole by intraperitoneal injection caused severe degeneration of OE. In further experiments olfactory turbinates were exposed to a range of concentrations (0-100 mM) of sodium carbonate, acetic acid and 3-methylindole for 4 h and ATP concentrations determined. Concentration-dependent decreases in ATP were observed for sodium carbonate and 3-methylindole, with EC(50) values estimated as 2.57 and 0.91 mM, respectively. Acetic acid only decreased ATP significantly at the 100-mM concentration. In summary, this in vitro model has predicted the nasal toxicity of several compounds, including both direct-acting agents (sodium carbonate, acetic acid) and one requiring metabolic activation (3-methylindole). However, the lack of airflow-dependent dosimetry, results in some lack of discrimination between the different regions of the nasal cavity and may make this model overly sensitive.

Histochemical localisation of carboxylesterase activity in rat and mouse oral cavity mucosa.

Vinyl acetate (VA) is widely used within the chemical industry, in the manufacture of polyvinyl alcohol, and as polyvinyl acetate emulsions in latex paints, adhesives, paper and paper board coatings. Chronic oral exposure of rodents to high concentrations of VA induces tumours within the oral cavity. Carboxylesterase-dependent hydrolysis of VA is thought to be critical in the development of nasal tumours following inhalation exposure of animals to VA. Therefore, carboxylesterase activity was determined histochemically in the oral cavities of male F344 rats and BDF mice in order to explore the potential role of carboxylesterase-dependent hydrolysis of VA in the development of oral tumours. Following fixation in 10% neutral buffered formalin heads were decalcified in neutral saturated EDTA, embedded in resin, sectioned at six levels (three each for the upper and lower jaws), and carboxylesterase activity revealed in the tissue using alpha-naphthyl butyrate as substrate. The localisation of carboxylesterase activity in freshly dissected rat oral tissue was compared to that of the resin sections and found to be identical, thus validating the decalcification process. A similar pattern of carboxylesterase activity was observed for the two species. Staining was low in areas surrounding the teeth, and medium/high in the buccal mucosa, the central/posterior upper palate and those regions of the lower jaw not proximal to the teeth. In general the intensity of staining was greater in sections from the rat compared to those from the mouse. By comparison, carboxylesterase activity was considerably higher in mouse nasal olfactory epithelium than in any of the oral tissues. Thus the mucosa of the oral cavity has the potential to hydrolyse VA to its metabolites, acetic acid and acetaldehyde, and the presence of carboxylesterases at this site is consistent with, and may be an important determining factor in, the development of oral cavity tumours following exposure to VA.

Regulation of rat olfactory glutathione S-transferase expression : Investigation of sex differences, induction, and ontogenesis

The glutathione S-transferases (GSTs) of rat olfactory epithelium have been characterised with regard to sex differences, induction, and developmental regulation, and compared to those of the liver. Olfactory cytosolic GST activity with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate was similar in both male and female animals, and there were no differences in subunit profile. Administration of trans-stilbene oxide and beta-naphthoflavone had no effect on olfactory GST activity with CDNB, although phenobarbitone treatment resulted in a small, but significant, increase in activity (130% compared to controls). HPLC analysis of subunit profiles indicated that all three agents induced olfactory subunit 1b and decreased subunit 6. The effect of age (3 to 84 days) on both cytosolic and microsomal CDNB activity was examined. In the liver, cytosolic activity was low at 3 days and climbed steadily to reach maximal levels around 28 days, but microsomal activity was relatively constant at all ages. Olfactory cytosolic activity was similar at all ages; microsomal activity was low until 21 days and then increased to reach a maximum at 56 days. Changes in individual cytosolic subunits were assessed by SDS-PAGE followed by immunoblotting. The significance of these results with regard to putative physiological roles for olfactory GSTs is discussed.