B. van Ommen

Toxicity of bis(tri-n-butyltin)oxide in the rat: II. Suppression of thymus-dependent immune responses and of parameters of nonspecific resistance after short-term exposure

To evaluate the functional significance of bis(tri-n-butyltin)oxide (TBTO)-induced thymus atrophy, lymphocyte depletion in spleen and lymph nodes, lymphopenia, and increased serum IgM and decreased IgG concentrations, in vivo and in vitro function studies were performed for specific and nonspecific resistance. Weaned male rats were fed diets containing 0, 20, or 80 mg TBTO/kg for a least 6 weeks. Regarding the thymus-dependent immunity, delayed-type hypersensitivity reactions to ovalbumin as well as tuberculin were significantly depressed at both dietary concentrations. Resistance to the nematode Trichinella spiralis was significantly suppressed as shown by a retarded expulsion of adult worms from the small intestine, increased counts of muscle larvae, reduced inflammatory reaction in parasitized musculature, and suppressed serum IgE titers. Also the secondary mercaptoethanol-resistant (presumably IgG) hemagglutinating antibody titer to sheep red blood cells was significantly reduced, while no significant alterations were found in IgM and IgG titers to T. spiralis, ovalbumin, and tetanus toxoid. TBTO exposure reduced the response of thymocytes in both treatment groups and of spleen cells in the 80-mg/kg group upon stimulation with T-cell mitogens and increased the response of spleen cells to B-cell mitogens. When calculated per whole spleen, the response to T-cell mitogens was strongly impaired but unaltered by B-cell mitogens. This difference can be explained by a relative increase of splenic B cells as a result of reduced numbers of T cells, as shown by cell surface marker analysis using monoclonal antibodies. Reduced splenic T-cell numbers appeared equally due to a decreased number of T helper and to T suppressor cells. From these data and from results of a time-sequence study in which effects of TBTO on cell count and cell viability of thymus, spleen, and bone marrow were investigated, it is concluded that TBTO-induced immunodeficiency was primarily due to its direct toxic action on thymocytes. When cultured in vitro in the presence of TBTO, viability of thymus and bone marrow cells was equally reduced, while after in vivo treatment viability of bone marrow cells was unaffected. Thus, the in vitro situation does not mimic the in vivo one. Concerning the nonspecific resistance. TBTO reduced macrophage function as shown by impaired splenic clearance of Listeria monocytogenes bacteria. From in vitro studies it is concluded that impaired in vivo splenic clearance was due to a reduction in both the number of adherent cells in the spleen and bacterial digestion on a cell for cell basis. The effect of TBTO on natural cell-mediated cytotoxicity was investigated in a 51Cr-release assay with YAC lymphoma target cells. In this test, the activity of natural killer (NK) cells in the spleen was significantly suppressed in the 80 mg/kg group while NK cell activity of peritoneal cells was unaltered. However, the activity of adherent peritoneal cells (cytotoxic macrophages) was significantly reduced in the 20 and 80 mg/kg groups. Finally, TBTO did not render rats more susceptible to the lethal effects of endotoxin. From the results it is concluded that low-dose feeding of TBTO suppresses thymus-dependent immune responses as well as parameters of the nonspecific resistance.

Ethacrynic acid and its glutathione conjugate as inhibitors of glutathione S-transferases.

1. The diuretic drug ethacrynic acid (EA) is a potent reversible inhibitor of rat and human glutathione S-transferases (GST), with I50-values (microM) of 4.6-6.0, 0.3-1.9 and 3.3-4.8 for alpha, mu and pi-class, respectively. 2. The reversible inhibition by the glutathione conjugate of EA is even stronger for alpha and mu-class, with I50-values (microM) of 0.8-2.8 and < 0.1-1.2, respectively, while the I50 for the pi-class is 11. 3. Inhibition of rat and human pi-class GST also occurs by covalent binding of ethacrynic acid. 14C-ethacrynic acid, 0.8 nmol EA per nmol pi-class GST could be incorporated, resulting in 65-93% inhibition of the catalytic activity. 4. Owing to the chemical nature of the covalent binding (Michael addition), this reaction should be reversible. Indeed, full restoration of the catalytic activity of GST P1-1 inactivated by covalently-bound EA was reached in about 125 h by incubation with an excess of glutathione. 5. EA has been used to inhibit GST in biological systems. The reversible covalent binding may very well play a role in the observed inhibition of GST by EA in vivo.

Glutathione S-transferase subunit induction patterns of Brussels sprouts, allyl isothiocyanate and goitrin in rat liver and small intestinal mucosa: a new approach for the identification of inducing xenobiotics.

Effects of Brussels sprouts (2.5-30%), allyl isothiocyanate (0.03 and 0.1%) and goitrin (0.02%), in the diet, on the glutathione S-transferase subunit pattern in the liver and small intestinal mucosa of male Fisher rats were investigated. A statistically significant linear relationship was found between the amount of Brussels sprouts in the diet and the induction of glutathione S-transferase subunits in two experiments. Increases in total activity of glutathione S-transferases towards 1-chloro-2,4-dinitrobenzene ranged from about 15% (2.5% Brussels sprouts in the diet) to 180% (30% Brussels sprouts in the diet) in the liver, and from 3% (2.5% Brussels sprouts) to 150% (30% Brussels sprouts) in the small intestinal mucosa. There were similar increases in the total amounts of glutathione S-transferase subunits. In the first experiment, when the average sinigrin and progoitrin levels found in the sprouts were 1835 and 415 mumol/kg, respectively, subunit induction patterns in both the liver and the small intestinal mucosa were very similar to the pattern observed after feeding allyl isothiocyanate. In the second experiment, when the average sinigrin level found in the sprouts was as low as the progoitrin level (both about 540 mumol/kg), a goitrin-like induction pattern was observed. The most pronounced difference between the glutathione S-transferase subunit induction patterns due to administration of allyl isothiocyanate and goitrin is the much stronger enhancement of subunit 2 by allyl isothiocyanate. The induction patterns of both experiments indicate that in Brussels sprouts at least two compounds, probably allyl isothiocyanate and goitrin, are responsible for the induction of glutathione S-transferases.

The microsomal metabolism of hexachlorobenzene. Origin of the covalent binding to protein.

The microsomal metabolism of hexachlorobenzene is studied, with special attention to the covalent binding to protein. The metabolites formed are pentachlorophenol and tetrachlorohydroquinone. In addition, a considerable amount of covalent binding to protein is detected (250 pmoles pentachlorophenol, 17 pmoles tetrachlorohydroquinone and 11 pmoles covalent binding in an incubation containing 50 mumoles of hexachlorobenzene). In order to establish the potential role of reductive dechlorination in the covalent binding, the anaerobic metabolism of hexachlorobenzene was investigated. At low oxygen concentrations no pentachlorobenzene was detected, and only very small amounts of pentachlorophenol as well as covalent binding, indicating a relationship between covalent binding and the microsomal oxidation of hexachlorobenzene. Incubations with 14C-pentachlorophenol at low concentrations showed that a conversion-dependent covalent binding occurs to the extent of 75 pmole binding per nmole pentachlorophenol. This is almost enough to account for the amount of label bound to protein observed in hexachlorobenzene incubations. This indicates that less than 10% of the covalent binding occurs during conversion of hexachlorobenzene to pentachlorophenol, and the remainder is produced during conversion of hexachlorobenzene to pentachlorophenol, and the remainder is produced during conversion of pentachlorophenol. The major product of microsomal oxidation of pentachlorophenol is tetrachlorohydroquinone, which is in redox-equilibrium with the corresponding semiquinone and quinone (chloranil). The covalent binding is inhibited by addition of ascorbic acid or glutathione to the hexachlorobenzene incubations. Ascorbic acid decreases the covalent binding with a simultaneous increase in formation of tetrachlorohydroquinone, probably due to a shift in the redox-equilibrium to the reduced side. Glutathione does not act as a reducing agent, since the inhibition of covalent binding is not accompanied by an increase in tetrachlorohydroquinone formation. Instead, glutathione reacts with chloranil, producing at least three stable products, probably in a Michael-type reaction. These results strongly indicate the involvement of chloranil or the semiquinone radical in the covalent binding during microsomal hexachlorobenzene metabolism.

The microsomal metabolism of pentachlorophenol and its covalent binding to protein and DNA

The microsomal metabolism of pentachlorophenol (PCP) was investigated, with special attention to the conversion dependent covalent binding to protein and DNA. The two metabolites detected were tetrachloro-1,2- and tetrachloro-1,4-hydroquinone. Microsomes from isosafrole (ISF)-induced rats were by far the most effective in catalyzing the reaction: the rate of conversion was increased 7-fold over control microsomes. All other inducers tested (hexachlorobenzene (HCB), phenobarbital (PB) and 3-methylcholanthrene (3MC) gave 2--3-fold increases over control. There are indications that the 1,2- and 1,4-isomers are produced in different ratios by various cytochrome P-450 isoenzymes: Microsomes from PB- and HCB-treated rats produced the tetrachloro-1,4- and tetrachloro-1,2-hydroquinone in a ratio of about 2, while microsomes from rats induced with 3 MC and ISF showed a ratio of about 1.3. When PCP was incubated with microsomes from rats treated with HCB, a mixed type inducer of P-450, the ratio between formation of the 1,4- and 1,2-isomers decreased with increasing concentration of PCP, suggesting the involvement of at least two P-450 isoenzymes with different Km-values. The overall apparent Km-value for HCB-microsomes was 13 microM both for the formation of the soluble metabolites and the covalent binding to microsomal protein, suggesting both stem from the same reaction. The covalent binding could be inhibited by ascorbic acid and this inhibition was accompanied by an increase in formation of tetrachlorohydroquinones (TCHQ). Although a large variation was observed in rates of conversion between microsomes treated with different (or no) inducers, the rate of covalent binding to microsomal protein was remarkably constant. A conversion-dependent covalent binding to DNA was observed in incubations with added DNA which was 0.2 times the amount of binding to protein (37 pmol/mg DNA).

Dietary n-6 and n-3 polyunsaturated fatty acids and colorectal carcinogenesis: results from cultured colon cells, animal models and human studies

During the past few decades, many studies have been conducted to evaluate the effects of n-6 and n-3 polyunsaturated fatty acids (PUFAs) on colorectal carcinogenesis. This report provides a brief overview of the recent studies that have been performed in cultured colon cells, animal models as well as of the population-based and short-term biomarker studies with humans. No differential effect between n-6 and n-3 PUFAs has been observed in vitro. Results from animal models indicate that n-6 PUFAs have a tumor enhancing effect, predominantly during the post-initiation phase. n-3 PUFAs may protect against colorectal carcinogenesis during both the initiation and post-initiation phase. Population-based human studies show little or no associations between n-6 or n-3 PUFA intake and colorectal cancer. Short-term biomarker studies in humans suggest though that fish oil (FO) supplementation with high amounts of n-3 PUFAs may protect against colorectal carcinogenesis and that n-6 PUFA supplementation may increase the risk.

Effect of inducers of cytochrome P-450 on the metabolism of [3-14C]coumarin by rat hepatic microsomes.

1. The metabolism of [3-14C]coumarin has been studied in rat hepatic microsomes and with two purified cytochrome P-450 isoenzymes. 2. [3-14C]Coumarin was converted by liver microsomes to several polar products including 3- and/or 5-hydroxycoumarin, omicron-hydroxyphenylacetic acid and a major unidentified novel coumarin metabolite. 3. [3-14C]Coumarin was also converted to reactive metabolite(s) as indicated by covalent binding to proteins, and by the depletion of reduced glutathione added to the microsomal incubations. 4. [3-14C]Coumarin metabolism to polar and covalently bound metabolites by rat liver microsomes was induced by pretreatment with phenobarbitone, 3-methylcholanthrene, beta-naphthoflavone, Aroclor 1254 and isosafrole; but not by dexamethasone or nafenopin. 5. The profile of [3-14C]coumarin metabolism to polar products was similar in control and pretreated liver microsomes and in incubations with purified cytochrome P450 IA1 and P450 IIB1 isoenzymes. 6. The results indicate that coumarin is a substrate for isoenzymes of the cytochrome P450 IA and P450 IIB subfamilies. The bioactivation of coumarin by rat hepatic microsomes is postulated to result in the formation of a coumarin 3,4-epoxide intermediate which may rearrange to 3-hydroxycoumarin, be further metabolized to a coumarin 3,4-dihydrodiol, or form a glutathione conjugate.

Glutathione S-transferase activity and isoenzyme composition in benign ovarian tumours, untreated malignant ovarian tumours, and malignant ovarian tumours after platinum/cyclophosphamide chemotherapy.

Glutathione S-transferase (GST) isoenzyme composition, isoenzyme quantities and enzymatic activity were investigated in benign (n = 4) ovarian tumours and malignant ovarian tumours, before (n = 20) and after (n = 16) chemotherapy. Enzymatic activity of GST in cytosols was measured by determining 1-chloro-2,4-dinitrobenzene conjugation with glutathione, cytosolic GST subunits were determined by wide pore reversed phase HPLC, using a S-hexylglutathione-agarose affinity column, and isoelectric focussing. Both GST activity and GST pi amount were not related to histopathologic type, differentiation grade, or tumour volume index in untreated malignant tumours. GST isoenzyme patterns were identical in benign tumours and malignant tumours before and after platinum/cyclophosphamide chemotherapy, while GST pi was the predominant transferase. Mean GST activity and GST pi amount were decreased (P < 0.05) in malignant ovarian tumours after platinum/cyclophosphamide chemotherapy compared to untreated ovarian malignant tumours. No relation was found in untreated ovarian tumours between GST pi amount and response to platinum/cyclophosphamide chemotherapy. Thus, within the limitations of the current study no arguments were found for a role of GST in in vivo drug resistance of malignant ovarian tumours to platinum/cyclophosphamide chemotherapy.

In vitro and in vivo reversible and irreversible inhibition of rat glutathione S-transferase isoenzymes by caffeic acid and its 2-S-glutathionyl conjugate

The reversible and irreversible inhibition of glutathione S-transferases (GST) by caffeic acid [3-(3,4-dihydroxyphenyl)-2-propenoic acid] was studied in vitro using purified rat isoenzymes, and in vivo in male Wistar (WU) rats. The concentrations of caffeic acid that inhibited reversibly 50% of the activity of different GST isoenzymes towards 1-chloro-2,4-dinitrobenzene (CDNB) (I50 values) were 58 (GST 4-4), 360 (GST 3-3) and 470 microM (GST 7-7), and higher than 640 microM for GST isoenzymes of the alpha class (GST 1-1 and 2-2). The major glutathione conjugate of caffeic acid, 2-S-glutathionylcaffeic acid (2-GSCA), was a much more potent reversible inhibitor of GST, with I50 values of 7.1 (GST 3-3), 13 (GST 1-1), 26 (GST 4-4), 36 (GST 7-7) and more than 125 microM (GST 2-2). On the other hand, caffeic acid was a much more efficient irreversible inhibitor of GST than 2-GSCA. In this respect, GST 7-7 was by far the most sensitive enzyme. The remaining activity towards CDNB (expressed as percentage of control) after incubating 1.25 microM-GST with 100 microM-caffeic acid for 6 hr at 37 degrees C was 34 (GST 2-2), 24 (GST 1-1), 23 (GST 4-4), 10 (GST 3-3) and 5% (GST 7-7). Almost no irreversible inhibition of GST 1-1 and 3-3 occurred during incubation with 2-GSCA. Incubation of caffeic acid with liver microsomes from dexamethasone-induced rats catalysed the oxidation of caffeic acid about 18 times more effectively as compared with the spontaneous oxidation, as determined by the formation of GSH conjugates from caffeic acid. In vivo, the effect of single oral doses of caffeic acid (50-500 mg/kg body weight) on the cytosolic GST activity towards CDNB was studied 18 hr after dosing in the liver, kidney and intestinal mucosa. A marginal but significant linear relationship was found between the amount of caffeic acid dosed and the irreversible inhibition of GST activity in the liver, with a maximum of about 14% inhibition in the highest dose group. This inhibition coincided with a small decrease in the mu-class GST subunits, which was only significant for GST subunit 4.

The oxidation of tetrachloro-1,4-hydroquinone by microsomes and purified cytochrome P-450b. Implications for covalent binding to protein and involvement of reactive oxygen species

The enzymatic oxidation of tetrachloro-1,4-hydroquinone (1,4-TCHQ), resulting in covalent binding to protein of tetrachloro-1,4-benzoquinone (1,4-TCBQ), was investigated, with special attention to the involvement of cytochrome P-450 and reactive oxygen species. 1,4-TCBQ itself reacted very rapidly and extensively with protein (58% of the 10 nmol added to 2 mg of protein, in a 5-min incubation). Ascorbic acid and glutathione prevented covalent binding of 1,4-TCBQ to protein, both when added directly and when formed from 1,4-TCHQ by microsomes. In microsomal incubations as well as in a reconstituted system containing purified cytochrome P-450b, 1,4-TCHQ oxidation and subsequent protein binding was shown to be completely dependent on NADPH. The reaction was to a large extent, but not completely, dependent on oxygen (83% decrease in binding under anaerobic conditions). Inhibition of cytochrome P-450 by metyrapone, which is also known to block the P-450-mediated formation of reactive oxygen species, gave a 80% decrease in binding, while the addition of superoxide dismutase prevented 75% of the covalent binding, almost the same amount as found in anerobic incubations. A large part of the conversion of 1,4-TCHQ to 1,4-TCBQ is apparently not catalyzed by cytochrome P-450 itself, but is mediated by superoxide anion formed by this enzyme. The involvement of this radical anion is also demonstrated by microsomal incubations without NADPH but including the xantine/xantine oxidase superoxide anion generating system. These incubations resulted in a 1.6-fold binding as compared to the binding in incubations with NADPH but without xantine/xantine oxidase. 1,4-TCHQ was shown to stimulate the oxidase activity of microsomal cytochrome P-450. It is thus not unlikely that 1,4-TCHQ enhances its own microsomal oxidation.