Mercury-induced H2O2 production and lipid peroxidation in vitro in rat kidney mitochondria.

Mercuric ion (Hg(II)) causes oxidative tissue damage in kidney cortical cells. We studied the in vitro effects of Hg(II) on hydrogen peroxide (H2O2) production by rat kidney mitochondria, a principal intracellular target of Hg(II). In mitochondria supplemented with a respiratory chain substrate (succinate or malate/glutamate) and an electron transport inhibitor (antimycin A (AA) or rotenone), Hg(II) (30 nmol/mg protein) increased H2O2 formation approximately 4-fold at the ubiquinone-cytochrome b region (AA-inhibited) and 2-fold at the NADH dehydrogenase region (rotenone-inhibited). Concomitantly, Hg(II) increased iron-dependent lipid peroxidation 3.5-fold at the NADH dehydrogenase region, but only by 25% at the ubiquinone-cytochrome b region. The mitochondrial concentration of reduced glutathione (GSH) decreased both with incubation time and Hg(II) concentration. Hg(II), at a concentration of 12 nmol/mg protein, caused almost complete depletion of measurable GSH in substrate-supplemented mitochondria after a 30-min incubation. In electron transport-inhibited mitochondria, Hg(II) caused greater depletion of GSH in rotenone-inhibited than in AA-inhibited mitochondria, consistent with the effects of Hg(II) on lipid peroxidation. These results suggest that Hg(II) at low concentrations depletes mitochondrial GSH and enhances H2O2 formation in kidney mitochondria under conditions of impaired respiratory chain electron transport. The increased H2O2 formation by Hg(II) may lead to oxidative tissue damage, such as lipid peroxidation, observed in mercury-induced nephrotoxicity.

Studies on Hg(II)-induced H2O2 formation and oxidative stress in vivo and in vitro in rat kidney mitochondria.

Studies were undertaken to investigate the principal actions underlying mercury-induced oxidative stress in the kidney. Mitochondria from kidneys of rats treated with HgCl2 (1.5 mg/kg i.p.) demonstrated a 2-fold increase in hydrogen peroxide (H2O2) formation for up to 6 hr following Hg(II) treatment using succinate as the electron transport chain substrate. No increase in H2O2 formation was observed when NAD-linked substrates (malate/glutamate) were used, suggesting that Hg(II) affects H2O2 formation principally at the ubiquinone-cytochrome b region of the mitochondrial respiratory chain in vivo. Together with increased H2O2 formation, mitochondrial glutathione (GSH) content was depleted by more than 50% following Hg(II) treatment, whereas formation of thiobarbiturate reactive substances (TBARS), indicative of mitochondrial lipid peroxidation, was increased by 68%. Studies in vivo revealed a significant concentration-related depolarization of the inner mitochondrial membrane following the addition of Hg(II) to mitochondria isolated from kidneys of untreated rats. This effect was accompanied by significantly increased H2O2 formation, GSH depletion and TBARS formation linked to both NADH dehydrogenase (rotenone-inhibited) and ubiquinone-cytochrome b (antimycin-inhibited) regions of the electron transport chain. Oxidation of pyridine nucleotides (NAD[P]H) was also observed in mitochondria incubated with Hg(II) in vitro. In further studies in vitro, the potential role of Ca2+ in Hg(II)-induced mitochondrial oxidative stress was investigated. Ca2+ alone (30-400 nmol/mg protein) produced no increase in H2O2 and only a slight increase in TBARS formation when incubated with kidney mitochondria isolated from untreated rats. However, Ca2+ significantly increased H2O2 and TBARS formation elicited by Hg(II) at the ubiquinone-cytochrome b region of the mitochondrial electron transport chain, whereas TBARS formation was decreased significantly when the Ca2+ uptake inhibitors, ruthenium red or [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), were included with Hg(II) in the reaction mixtures. These findings support the view that Hg(II) causes depolarization of the mitochondrial inner membrane with consequent increased H2O2 formation. These events, coupled with Hg(II)-mediated GSH depletion and pyridine nucleotide oxidation, create an oxidant stress condition characterized by increased susceptibility of mitochondrial membranes to iron-dependent lipid peroxidation (TBARS formation). Since increased H2O2 formation, GSH depletion and lipid peroxidation were also observed in vivo following Hg(II) treatment, these events may underlie oxidative tissue damage caused by mercury compounds. Moreover, Hg(II)-induced alterations in mitochondrial Ca2+ homeostasis may exacerbate Hg(II)-induced oxidative stress in kidney cells.

Interactions between methylsulfonyl PCBs and the glucocorticoid receptor.

Persistent polychlorinated biphenyl (PCB) metabolites were studied with respect to their interaction with the human glucocorticoid receptor (GR). 3-Methylsulphonyl-2,5,6,2',4',5'-hexachlorobiphenyl (3-MeSO2-CB149) was shown to compete with 3H-dexamethasone for binding to the GR, with an IC50 (concentration that inhibits 50%) of approximately 1 microM. Using GRAF cells expressing human GR, glucocorticoid responsive element, and a reporter enzyme, we demonstrated that 3-MeSO2-CB149 functionally acts as an antagonist at the GR (IC50 = 2.7 microM). In accordance with the receptor binding, the antagonism mainly appeared to be of a competitive nature. When studying the competitive binding of 24 methylsulfonyl PCBs (relative to dexamethasone) to GR from mouse liver cytosol, seven compounds had a higher affinity to GR than 3-MeSO2-CB149. Structure-activity relationship studies indicated that the presence of three chlorine atoms in the ortho-position and chlorine and methyl sulfone groups on either end of the molecule (4 and 4'-position) increased the affinity to GR. The relevance of this finding for human health is not known, but PCB methyl sulfones are ubiquitous pollutants present in mother's milk. The results stress the need for studying endocrine disruptors that affect hormonal systems other than sex and thyroidogenic hormones.

Irreversible binding and adrenocorticolytic activity of the DDT metabolite 3-methylsulfonyl-DDE examined in tissue-slice culture.

The persistent adrenocorticolytic DDT metabolite 3-methylsulfonyl-DDE (MeSO(2)-DDE) was originally identified in Baltic grey seals, a population suffering from adrenocortical hyperplasia. In mice, MeSO(2)-DDE induces mitochondrial degeneration and cellular necrosis in the adrenal zona fasciculata. In this study, we used precision-cut tissue slice culture to examine local CYP11B1-catalyzed irreversible binding of MeSO(2)-DDE in the murine adrenal cortex. We also examined effects on steroid hormone secretion, histology, and ultrastructure. As determined by microautoradiography, selective binding occurred in zona fasciculata of slices exposed to MeSO(2)-[(14)C]-DDE. Quantification of binding by phosphorautoradiography revealed a 3-fold reduction of binding in slices co-exposed to the CYP11B1 inhibitor metyrapone. As measured by HPLC, corticosterone and 11-deoxycorticosterone secretion to the medium increased linearly for at least 24 hr. Addition of the ACTH analog tetracosactide caused an 8-fold increase in corticosterone secretion. Addition of metyrapone reduced corticosterone secretion 4-fold. Exposure of slices to MeSO(2)-DDE (50 microM) reduced the rate of corticosterone secretion by 90% after 24 hr of incubation. As determined by electron microscopy, vacuolated mitochondria were present in zona fasciculata of slices exposed to MeSO(2)-DDE (50 microM) for 24 hr. Our findings show that all effects of MeSO(2)-DDE previously reported in vivo could be reproduced in adrenal slice culture ex vivo. This test system allows analysis of zone-specific irreversible binding and effects on steroid hormone secretion and target cell ultrastructure. We propose adrenal slice culture as a simple ex vivo test system with which to examine the adrenocorticolytic activity of xenobiotics in human and wild animal tissue.

o,p'-DDD in the mouse lung: selective uptake, covalent binding and effect on drug metabolism.

By means of autoradiography a high and selective accumulation was observed in the lung alveolar region of C57Bl mice injected with o,p'-[14C]DDD. Exhaustive extraction of lung tissue showed that a large fraction of the radioactivity was covalently bound to protein. Covalent binding in liver was 20-30 times lower and represented a smaller fraction of the total radioactivity present in this tissue. Formation of a cytochrome P-450 catalysed reactive metabolite in lung and liver was indicated by a decreased covalent binding in these tissues in mice pretreated with metyrapone. Both beta-naphthoflavone (beta NF) and phenobarbital (PB) pretreatment decreased binding of o,p'-DDD in lung tissue, while binding in the liver was induced by PB but remained unaffected by beta NF. Pretreatment with high doses of o,p'-DDD and p,p'-DDT gave a significantly decreased binding of o,p'-[14C]DDD in lung, whereas binding in liver remained unchanged. Conjugation with glutathione does not appear to be a major inactivation pathway for the reactive lung metabolite, since a high dose of o,p'-DDD did not deplete non-protein thiols (NPSH) in lung tissue. Pretreatment with o,p'-DDD decreased the N-demethylation of [dimethyl-14C]aminopyrine in both lung and liver in a dose-dependent manner, suggesting that the drug-metabolizing enzyme system may be a target for o,p'-DDD in vivo.

Metabolic activation and toxicity of a DDT-metabolite, 3-methylsulphonyl-DDE, in the adrenal zona fasciculata in mice.

Whole-body autoradiography of 14C-labelled 3-methylsulphonyl-DDE (3-MeSO2-DDE) in female C57BL mice revealed a heavy accumulation in the adrenal cortex. Fairly high radioactivity appeared in the nasal mucosa and fat, while the labelling of the liver was intermediate. The adrenal radioactivity remained largely unextracted in tissue-sections treated with organic solvents. In the liver and intestinal contents the radioactivity was partly extracted, whereas in all other tissues almost completely extracted. According to light microscopic autoradiography, the tissue-bound adrenal radioactivity was confined to the zona fasciculata, leaving the other adrenal zones devoid of bound material. Incubation of 3-MeSO2-DDE with adrenal tissue (300 X g supernatant) revealed a dose- and time-dependent covalent binding to protein and formation of water-soluble metabolites. The cytochrome P-450 inhibitors metyrapone and carbon monoxide inhibited both covalent binding and polar metabolite formation. Addition of reduced glutathione decreased binding, while polar metabolite formation was increased. Histopathological examination of adrenals from 3-MeSO2-DDE-treated mice revealed extensive vacuolation and necrosis of the zona fasciculata 1-12 days after single doses down to 25 mg/kg. Degenerative changes were observed at 12.5 mg/kg. In contrast to 3-MeSO2-DDE, 14C-labelled 3,3'-bis(methylsulphonyl)-DDE was not accumulated in the adrenal cortex. 3-MeSO2-DDE is thus a persistent environmental pollutant with a unique ability to produce acute toxicity subsequent to metabolic activation in a mammalian tissue.

In vitro macromolecular binding of 2-(2-chlorophenyl)-2-(4-chlorophenyl)-1,1-dichloroethane (o,p'-DDD) in the mouse lung and liver.

The activation and covalent binding of 14C-labelled 2-(2-chlorophenyl)-2-(4-chlorophenyl)-1,1-dichloroethane (o,p'-DDD) in mouse lung and liver S-9 preparations were examined in vitro. These results showed an oxidative cytochrome P-450 mediated transformation of o,p'-DDD to metabolite(s) that bind covalently to proteins, phospholipids and to added naked DNA in both lung and liver. The apparent Km-values for the covalent binding of o,p'-DDD to protein were 0.25 microM and 3.30 microM in lung and liver, respectively. Addition of glutathione to the incubation medium decreased the binding of o,p'-DDD more efficiently in the liver than in the lung. Thus, the selective lung binding of o,p'-DDD previously observed in vivo seems to result from an in situ activation. The tissue selectivity in vivo is suggested to be due to the low apparent Km in the lung favouring bioactivation at low, ecotoxicologically relevant doses, as well as to a less pronounced protection by glutathione in the lung.

In vitro bioactivation of the environmental pollutant 3-methylsulphonyl-2, 2-bis(4-chlorophenyl)-1,1-dichloroethene in the human adrenal gland.

The metabolic activation and covalent binding of 3-methylsulphonyl-2,2-bis (4-chlorophenyl)-1,1-dichloroethene (MeSO2-(14C)DDE)[correction of (14C)DDD)] were studied in vitro in the human adrenal gland. The mitochondrial bioactivation was twice as high as the microsomal, thus the study was focused on the mitochondria. The irreversible binding was time and protein dependent with apparent Km and Vmax values of 1.4 microM and 275 pmol/mg protein/h. As a comparison the activation of 2-(2-chlorophenyl)-2(4-chlorophenyl)-1,1-dichloroethane (o,p'-DDD), a well-known adrenolytic compound in humans, was studied. The irreversible binding of both these DDT metabolites was inhibited by metyrapone, indicating the involvement of cytochrome P-450. Addition of reduced glutathione (GSH) to the incubation medium decreased the irreversible binding of MeSO2-DDE significantly whereas the binding of o,p'-DDD was only slightly affected. The above findings suggest that MeSO2-DDE may be toxic to the human adrenal gland, although further study is necessary to assess this potential.

Decreased pulmonary drug metabolism in mice treated with the PCB metabolite 4-methylsulphonyl-2,2',5,5'-tetrachlorobiphenyl.

4-Methylsulphonyl-2,2',5,5'-tetrachlorobiphenyl (4-MeSO2-TCB) is a major polychlorinated biphenyl (PCB) metabolite present in lung tissue of PCB-exposed human subjects. After treatment of mice with 4-MeSO2-TCB (100 mg/kg), the pulmonary N-demethylation of aminopyrine in vitro was significantly decreased, while hepatic N-demethylation was concomitantly increased, as compared to tissue from control mice. Treatment of mice with 4-MeSO2-TCB also decreased the in vivo pulmonary covalent binding of o,p'-DDD, while the in vivo hepatic covalent binding was increased. The results indicate that 4-MeSO2-TCB inhibits or represses a cytochrome P-450-dependent enzyme activity in the mouse lung, while in contrast this activity is induced in the mouse liver.

Covalent binding of o,p'-DDD in rabbit lung and isolated rabbit lung cells.

The irreversible binding of o,p'-DDD was examined in isolated lung cells, in lung microsomes and in vivo in male New Zealand White rabbits. Non-ciliated bronchiolar (Clara) cells had the highest capacity to bind o,p'-DDD, followed by alveolar type II cells. A fraction of mixed unidentified lung cells was also able to bind o,p'-DDD while no binding was observed in alveolar macrophages. The activation of o,p'-DDD was shown to be mediated by cytochrome P-450 in both lung microsomes and isolated lung cells. In vivo, the binding was preferentially localized in the lung alveolar and bronchiolar regions. The binding of o,p'-DDD observed in vivo may thus be caused by the capacity of several cell types to activate o,p'-DDD.

Adrenocortical toxicity of 3-methylsulphonyl-DDE; 3: Studies in fetal and suckling mice.

Irreversible binding and toxicity of the DDT metabolite 3-methylsulphonyl-DDE (MeSO2-DDE) were examined in fetuses and suckling pups following administration to pregnant or lactating C57Bl mice. Tape-section autoradiography showed a high and tissue-specific accumulation and binding of MeSO2-DDE-14C-derived radioactivity in the late gestational fetal adrenal cortex. According to microautoradiography an irreversibly bound residue was confined to the zona fasciculata. Similarly, there was a high concentration of irreversibly bound 14C-labelled material in the adrenal zona fasciculata of suckling pups. Intraperitoneal injection of MeSO2-DDE-14C to lactating mice resulted in higher concentrations of radioactivity in the liver and stomach contents (milk) of the suckling pups than in the maternal liver. This treatment also resulted in a higher level of radioactivity in the adrenals of the pups than in the maternal adrenals, both at a subtoxic and at a toxic dose. Histopathologic examination of adrenals from suckling pups revealed extensive vacuolation and necrosis of the zona fasciculata 2 days following a single dose of MeSO2-DDE (25 mg/kg) to the dam. In the fetal adrenal zona fasciculata, slight degenerative changes were observed following a maternal dose of 50 mg/kg. In conclusion, the study shows that MeSO2-DDE is a highly tissue-specific toxicant to the fetal and postnatal adrenal zona fasciculata in mice. Based on the present data and on previous results in adult mice, we propose that a tissue-specific activation to a reactive metabolite in the fetal and postnatal adrenal cortex is mediated by cytochrome P-450 (11 beta).

Effects of 3-MeSO2-DDE and some CYP inhibitors on glucocorticoid steroidogenesis in the H295R human adrenocortical carcinoma cell line.

The formation of steroids in the H295R human adrenocortical carcinoma cell line was analysed by HPLC or RIA, and based on these data the apparent catalytic activities of CYP11A, CYP17, CYP21 and CYP11B1 in this cell line were calculated. The environmental pollutant 3-methylsulfonyl-DDE (3-MeSO2-DDE) and the cytochrome P450 (CYP) inhibitors ketoconazole, metyrapone and aminoglutethimide were studied for their effects on the steroid formation. Metyrapone (IC50) of 1 microM) and 3-MeSO2-DDE (10 microM: 66 +/- 10% of control) were found to inhibit the apparent CYP11B1 activity. Ketoconazole inhibited all enzymes examined with the greatest effects on CYP11B1 (IC50) of 2.5 microM). Aminoglutethimide was examined only for effects on CYP11A activity and was shown to inhibit pregnenolone formation (20 microM: 61 +/- 4% of control). The possibility of studying all CYP enzymes in the corticosteroidogenesis makes this cell line a valuable test system to examine effects of chemicals, such as suspected endocrine disruptors, on the human glucocorticoid hormone synthesis. The inhibition of cortisol formation by 3-MeSO2-DDE supports an interaction with the active site of CYP11B1, as previously reported in mouse adrenocortical Y1 cells. In mice, this interaction led to metabolic activation and a high adrenotoxicity of 3-MeSO2-DDE. Therefore studies on the adrenotoxicity of 3-MeSO2-DDE in humans are needed.

Reproductive toxicity in mink (Mustela vison) chronically exposed to environmentally relevant polychlorinated biphenyl concentrations.

Female mink were exposed to a technical polychlorinated biphenyl (PCB) preparation (Clophen A50 [A50]; 0.1 or 0.3 mg/animal/d), one fraction of A50 containing the non- and mono-ortho-chlorinated congeners (0-1-ortho-chlorobiphenyls [CBs]), another fraction of A50 containing the congeners with two to four ortho-chlorines (2-4-ortho-CBs), or an organic extract from Baltic gray seal blubber. The animals were exposed for 18 months, including two reproduction seasons. Among the animals given the highest dose of A50, the whelping frequency was reduced in the second reproductive season, and all kits died within 24 h of birth. Reproduction was also impaired by the lower dose of A50. Daily exposure to the 0-1-ortho-CBs separated from 0.3 mg A50 severely reduced kit survival. Reproduction was not significantly impaired by daily exposure to the 2-4-ortho-CBs separated from 0.3 mg A50 or by exposure to the blubber extract. We conclude that the reproductive toxicity in chronically PCB-exposed mink is caused by the aryl hydrocarbon (Ah) receptor agonists. The lowest-observed-effect level for reproductive impairment was 2.4 ng 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents (TEQs) per kilogram body weight and day (22 pg TEQs/g feed). Ethoxyresorufin-O-dealkylase (EROD) was strongly induced by the 0-1-ortho-CBs and pentoxyresorufin-O-dealkylase by the 2-4-ortho-CBs. High EROD activity was correlated with low kit production, and consequently EROD may serve as a marker for reproductive toxicity by Ah receptor agonists in mink.

Novel involvement of a mitochondrial steroid hydroxylase (P450c11) in xenobiotic metabolism.

Adrenocortical mitochondrial cytochrome P450 isozymes of the Cyp11 family normally synthesize steroids with a very strict substrate specificity. However, for the first time, P450c11 was additionally shown to metabolize and bioactivate the adrenotoxic environmental pollutant 3-methylsulfonyl-2,2-bis(4-chlorophenyl)-1,1-dichloroethene (MeSO2-DDE). This conclusion is based on a striking correlation between inductions of MeSO2-DDE and deoxycorticosterone metabolism by forskolin in the adrenocortical cell lines Y1 and Kin-8, inhibition of P450c11-dependent activities in Y1 cells by MeSO2-DDE, and metabolism of MeSO2-DDE by non-steroidogenic COS cells after transfection with a cDNA encoding P450c11. The interaction between xenobiotics and glucocorticoid synthesis should focus more attention to xenobiotic-induced hormonal disturbances.

Covalent binding of four DDD isomers in the mouse lung: lack of structure specificity.

Previous studies have shown that o,p'-DDD is activated and covalently bound in the mouse lung. In order to examine the structure dependency of the selective lung binding, the 14C-labelled DDD isomers p,p'-DDD, m,p'-DDD and o,m'-DDD were injected intravenously into female C57B1 mice and covalent binding was measured. Autoradiography of solvent-extracted tape-sections showed that all isomers were selectively and covalently bound in the lung alveolar region. As determined by exhaustive extraction of homogenized tissue, maximal binding was observed 4 hr after injection, although the lung/liver concentration ratio increased for 12 days. Covalent protein binding was also observed in vitro, implying that the activation of DDD to a reactive metabolite takes place in the target organ. Since the aryl-chlorine substitution pattern did not change the selective lung binding, bioactivation of DDD may take place at the ethane side-chain.

Structure-activity relationship for inhibition of CYP11B1-dependent glucocorticoid synthesis in Y1 cells by aryl methyl sulfones.

The effects of xenobiotics on CYP11B1-dependent corticosterone synthesis (11 beta-hydroxylase) in mouse adrenocortical Y1 cells were studied. 3-Methylsulfonyl-2,2-bis(4-chlorophenyl)-1,1-dichloroethene (MeSO2-DDE) and some methylsulfonyl polychlorinated biphenyls (MeSO2-PCB) inhibited the corticosterone synthesis, whereas PCBs or DDE did not. This indicates a crucial role of the methyl sulfone group for this inhibitory effect. Kinetic analyses of MeSO2-DDE and the two most potent MeSO2-PCBs were conducted using Lineweaver-Burk double-reciprocal plots. The data showed a competitive inhibition of CYP11B1 by the compounds, with apparent inhibitory constants (Ki) of 1.6, 4.6, and 6.7 microM for MeSO2-DDE, 4-MeSO2-2,3,6,4'-tetrachlorobiphenyl, and 4-MeSO2-2,3,6,3',4'-pentachlorobiphenyl, respectively. For comparison, the substrate K(m) was 3.5 microM in the cells, and metyrapone and ketoconazole had apparent Ki-values of 0.8 and 0.04 microM, respectively. In contrast to all previously known inhibitors of CYP11B1, the aryl methyl sulfones are the first examples of CYP11B1 inhibitors not being heterocyclic amines or steroids. The aryl methyl sulfones are widespread environmental pollutants and their inhibition of CYP11B1 constitutes another potential mechanism for endocrine disruption. Their influence on the synthesis of adrenocortical hormones thus merits further interest.

Effect of o,p'-DDD on the in vivo incorporation of 3H-thymidine into DNA: evidence for induced cell proliferation in the mouse lung.

The effects of o,p'-DDD on the DNA synthesis in the C57Bl mouse lung and liver were studied. As determined by 3H-thymidine incorporation into DNA, a selective increase in the lung DNA synthesis (+59%) was observed 2 days after a single intraperitoneal injection of 100 mg/kg o,p'-DDD. Microautoradiography showed that the incorporated 3H-thymidine was confined to a restricted number of heavily labelled cells, presumably proliferating type II cells. At the most, a 9 times higher rate of cell proliferation was observed in the lung 4 days after an intraperitoneal injection of 500 mg/kg o,p'-DDD. Using mouse lung or liver S-9 as activating system, no mutagenic activity of o,p'-DDD was detected in the Ames test. The induced cell proliferation may indicate a tissue-selective promoter activity of o,p'-DDD in the mouse lung.

Reactivity of Hg(II) with superoxide: evidence for the catalytic dismutation of superoxide by Hg(II).

Mercuric ion, a well-known nephrotoxin, promotes oxidative tissue damage to kidney cells. One principal toxic action of Hg(II) is the disruption of mitochondrial functions, although the exact significance of this effect with regard to Hg(II) toxicity is poorly understood. In studies of the effects of Hg(II) on superoxide (O2-) and hydrogen peroxide (H2O2) production by rat kidney mitochondria, Hg(II) (1-6 microM), in the presence of antimycin A, caused a concentration-dependent increase (up to fivefold) in mitochondrial H2O2 production but an apparent decrease in mitochondrial O2- production. Hg(II) also inhibited O(2-)-dependent cytochrome c reduction (IC50 approximately 2-3 microM) when O2- was produced from xanthine oxidase. In contrast, Hg(I) did not react with O2- in either system, suggesting little involvement of Hg(I) in the apparent dismutation of O2- by Hg(II). Hg(II) also inhibited the reactions of KO2 (i.e., O2-) with hemin or horseradish peroxidase dissolved in dimethyl sulfoxide (DMSO). Finally, a combination of Hg(II) and KO2 in DMSO resulted in a stable UV absorbance spectrum [currently assigned Hg(II)-peroxide] distinct from either Hg(II) or KO2. These results suggest that Hg(II), despite possessing little redox activity, enhances the rate of O2- dismutation, leading to increased production of H2O2 by renal mitochondria. This property of Hg(II) may contribute to the oxidative tissue-damaging properties of mercury compounds.

2,2',4,5,5'-Pentachlorobiphenyl: comparative metabolism in mink (Mustela vison) and mouse.

The metabolism of 14C-labeled 2,2',4,5,5'-pentachlorobiphenyl was studied in mink (Mustela vison) and, for comparison in the mouse (C57Bl). Both species were dosed orally and kept in metabolism cages for 5 days. Distribution in tissues and excretion rate were determined radiometrically, and metabolites were analyzed by GC/MS. Within 5 days, the mink excreted 17% of the dose, and the mouse excreted 74%, mainly via the feces. For both species, the excreted radioactivity consisted primarily of metabolites, a large proportion of which were covalently bound to macromolecules and to lipids. A smaller proportion consisted of water-soluble metabolites. Phenolic and, in trace amounts, methylsulfonyl metabolites were also excreted. In the mink, a salivary gland in the neck region demonstrated the highest concentration of radiolabeled material of all tissues examined (3 times higher than in adipose tissue and liver on a lipid weight basis). The radioactive material in the salivary gland consisted of the parent compound and phenolic and methylsulfonyl metabolites. In the mouse, the highest concentration of 14C was found in the lund and consisted of 4-methylsulfonyl-2,2',4',5,5'-pentachlorobiphenyl. Hydroxylated and methylsulfonyl metabolites were retained to various degrees in tissues of both species. The mink, but not the mouse, also formed metabolites that were hydroxylated in the trichlorinated phenyl ring.

Adrenocorticolytic DDT-metabolites: studies in mink, Mustela vison and otter, Lutra lutra.

: The irreversible binding and toxicity of the DDT metabolites p,p'-DDD and o,p-DDD in the adrenal cortex of female mink Mustela vison were studied. Histological examination of adrenals from mink given a single i.p. injection of p,p'-DDD or o,p'-DDD (125 mg per kg body weight) showed vacuolation, necrosis and focal bleedings in the zonae fasciculata and reticularis. Autoradiograms of solvent-extracted tissue sections of minks given a single i.v. injection of p,p'-[(14)C]DDD (0.7 mg per kg body weight) revealed a high level of irreversibly bound radio-activity in the adrenal cortex. Microautoradiography showed that the irreversibly bound radio-activity was confined to the zonae fasciculata and reticularis. Incubation of p,p'-[(14)C]DDD and o,p'-[(14)C]DDD with mink or otter, Lutra lutra, adrenal homogenate (300 x g supernatant) resulted in an irreversible binding of radioactivity to protein from both species. The irreversible protein binding of the DDD isomers in mink and otter was decreased by addition of the cytochrome P450 inhibitors metyrapone and carbon monoxide, indicating a cytochrome P450 dependent metabolic activation. In contrast, 3-methylsulfonyl-[(14)C]DDE, a potent adrenocortical toxicant in mice, does not appear to be metabolized to a reactive metabolite in the adrenal cortex of mink or otter. In conclusion, both p,p'-DDD and o,p'-DDD are toxic to the mink adrenal zona fasciculata and reticularis following activation in situ to reactive, tissue-binding metabolites. The results suggest that p,p'-DDD and o,p'-DDD are adrenocortical toxicants also in otter. The involvement of environmental pollutants in the generation of the adrenocortical hyperplasia observed among Baltic seals is discussed.