Joseph W. DePierre

Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver.

Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver have been investigated. After perfusing the lung to remove contaminating blood, this organ was found to have an apparent concentration of glutathione (2mM) which is approx. 20% of that found in the liver. Both organs contain very low levels of glutathione disulfide. Neither phenobarbital nor methylcholanthrene had a significant effect on the levels of reduced glutathione in lung and liver. In addition, the activities of some glutathione-metabolizing enzymes--glutathione reductase and glutathione S-transferase activity assayed with four different substrates--were observed to be 5-to 60-fold lower in lung tissue than in the liver.

Microsomal Glutathione S‐Transferase

Rat liver microsomal glutathione S-transferase was activated with N-ethylmaleimide, solubilized with Triton X-100, and purified by chromatography on hydroxyapatite and CM-Sepharose. A 36-fold purification resulted in a 36% yield, indicating that the glutathione S-transferase accounts for 2.5-3% of the original microsomal protein. The purified protein moved as a band with an apparent molecular weight of 14 000 on sodium dodecyl sulphate gel electrophoresis and appeared to be nearly homogeneous. The complex formed between the purified microsomal glutathione S-transferase and Triton X-100 has a sedimentation coefficient of 3.2 S, a partial specific volume of 0.844 cm3/g, and a Stokes radius of 5.5 nm. The complex has a molecular weight of 127 000 and contains three or four polypeptide chains and 112-134 detergent molecules. Antibodies directed against soluble glutathione S-transferases A, B and C do not react with the purified microsomal enzyme. This finding, together with differences in molecular weight and substrate specificity, demonstrate that the microsomal glutathione S-transferase is an enzyme distinct from the cytosolic glutathione S-transferases.

Tricyclic antidepressants inhibit IL-6, IL-1β and TNF-α release in human blood monocytes and IL-2 and interferon-γ in T cells

Tricyclic antidepressants (TCAs) are widely used in treating depressive disorders. It has been demonstrated that, for instance, IL-1 beta and IL-6 inhibit the HPA axis, which plays a role in the development of depressions. Therefore. we were interested in investigating how TCAs influence cytokine release by T lymphocytes and monocytes respectively. Cells were incubated with either 5 microM clomipramine, 15 microM imipramine or 20 microM citalopram. IL-2 release was suppressed to 60% of the control values by clomipramine and imipramine (p = 0.001; p = 0.000), but citalopram was found to cause a much weaker inhibition (only 18%) (p = 0.16). INF-gamma release was affected to a lower degree than IL-2 release, and imipramine (34%) (p = 0.054) was more potent than clomipramine (24%) (p = 0.16) and citalopram (12%) (p = 0.059) in this case. Monocytes incubated with TCA for 4 h exhibited only limited inhibition of IL-1 beta and IL-6 release, i.e., 6-25% for all three compounds. The corresponding value for TNF-alpha release was 20-45% inhibition, with citalopram being the weakest inhibitor. After 10 h of monocytes to LPS exposure, all three compounds exerted a strong inhibition of IL-1 beta and TNF-alpha release, i.e., 60-70% with p-values below 0.012 for all of them. However the inhibition of IL-6 release was less than 35%. Citalopram was equality as potent as imipramine and clomipramine in inhibiting IL-6 release after long-term exposure of monocytes to LPS. All three TCAs elevated intracellular cAMP concentrations significantly in T lymphocytes and monocytes (p < 0.001).

A reliable, sensitive, and convenient radioactive assay for benzpyrene monooxygenase

Abstract A new radioactive assay for benzpyrene monooxygenase has been developed, characterized, and compared to the fluorescent assay generally employed. The radioactive assay is based on a single extraction which effectively separates metabolites from remaining substrate. This assay is linear within reasonable ranges of time and protein concentration and responds as expected to inhibitors and an inducer of benzpyrene monooxygenase activity. The activity measured with the present assay is about twice that measured with the fluorescent assay. Furthermore, the radioactive assay can be scaled down so that it is at least as sensitive as the fluorescent assay.

Measurement of cytochrome P-450 in the presence of large amounts of contaminating hemoglobin and methemoglobin

Abstract A spectrophotometric method for assaying cytochrome P -450 in microsomes contaminated with large amounts of hemoglobin and methemoglobin has been developed. This method is based on the difference spectrum of dithionite-reduced microsomes minus nonreduced microsomes when both sample and reference have been bubbled with carbon monoxide. Because a significant amount of the contaminating hemoglobin is present in the oxidized form, the CO-hemoglobin peak is not balanced when reducing agent is added to the sample alone. It is therefore necessary to add 0.25 m m ascorbic acid and 2.5 μ m PES (phenazine ethosulfate), which reduces methemoglobin without reducing cytochrome P -450, to both cuvettes.

Activation of microsomal glutathione S-transferase activity by sulfhydryl reagents.

Abstract Rat liver microsomes exhibit glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene as the second substrate. This activity can be stimulated 8-fold by treatment of the microsomes with N-ethylmaleimide and 4-fold with iodoacetamide. The corresponding glutathione S-transferase activity of the supernatant fraction is not affected by such treatment. These findings suggest that rat liver microsomes contain glutathione S-transferase distinct from those found in the cytoplasmic and that the microsomal transferase can be activated by modification of microsomal sulfhydryl group(s).

The distribution of microsomal glutathione transferase among different organelles, different organs, and different organisms

In the present study we have used both enzyme assay with 1-chloro-2,4-dinitrobenzene as substrate and immunochemical quantitation to examine the distribution of microsomal glutathione transferase in different organelles, in different organs, and in different organisms. This enzyme was found to constitute 3% and 5%, respectively, of the total protein recovered in the microsomal and outer mitochondrial membrane fractions from rat liver. Microsomal glutathione transferase present in other subcellular fractions can be accounted for by contamination by the endoplasmic reticulum. In contrast to the situation with rat liver microsomes the glutathione transferase activities of microsomes from extrahepatic tissues of this same animal could not be activated by treatment with N-ethylmaleimide. Nonetheless, significant albeit low levels of a protein with the same molecular weight and immunochemical properties as the rat liver enzyme could be detected in microsomes from several extrahepatic tissues, notably the intestine, the adrenal, and the testis. Of those mammals for which fresh liver could be obtained, all demonstrated N-ethylmaleimide-activatable glutathione transferase activity in their liver microsomes. On the other hand, representatives for fish, birds, and amphibia did not demonstrate such activatable transferase activity in their liver microsomes. Toad was the only species that had a notable (twofold) sex difference in their level of hepatic microsomal glutathione transferase activity.

Potent suppression of the adaptive immune response in mice upon dietary exposure to the potent peroxisome proliferator, perfluorooctanoic acid

In a previous investigation, we demonstrated that severe thymus and spleen atrophy occurs in mice upon dietary exposure to several potent peroxisome proliferators (PPs). In the present investigation, the effects of the potent PP perfluorooctanoic acid (PFOA) on the adaptive immunity of mice was evaluated both in vivo and ex vivo. The in vivo immune response examined involved immunization of mice with horse red blood cells (HRBCs), displaying T-cell-dependent antigens after pre-treatment with a PFOA-containing diet for 10 days. Subsequent quantitation of the primary humoral response was performed employing both the plaque-forming cell (PFC) assay and determination of the antibody titer by ELISA. The results clearly demonstrate that oral administration of PFOA prevents both the increases in plaque formations by anti-IgM and -IgG and in serum levels of IgM and IgG normally evoked by such immunization. Ex vivo spleen cells proliferation (assayed as incorporation of 3H-thymidine) in response to both T- and B-cell activators was attenuated by dietary treatment with PFOA, although the analogous in vitro treatment of mouse spleen cells with this same compound had no such effects. Thus, the relatively metabolically inert PP PFOA may exert adaptive immunosuppression in mice by an indirect mechanism. The possible relevance of this immunosuppression to the alterations in plasma lipids caused by PPs is discussed.

Effects of peroxisome proliferators on the thymus and spleen of mice

The effects of peroxisome proliferators on the immune system of male C57Bl/6 mice have been investigated. Significant atrophy of the thymus and spleen was observed in animals treated with potent peroxisome proliferators (e.g. perfluorooctanoic acid (PFOA), di(2‐ethylhexyl)phthalate (DEHP), Wy‐14 643 and nafenopin), whereas the effects of a moderate peroxisome proliferator (i.e. acetylsalicylic acid (ASA)) were relatively weak. The time course of thymic and splenic atrophy caused by PFOA was found to resemble the time course of the increase in liver weight and of peroxisome proliferation. Analysis of the numbers and phenotypes of thymocytes and splenocytes from PFOA‐treated mice revealed the following: (i) the numbers of thymocytes and splenocytes were decreased > 90% and about 50%, respectively, by PFOA treatment; (ii) although all populations of thymocytes were decreased, the immature CD4+CD8+ population was decreased most dramatically; (iii) the numbers of both T and B cells in the spleen were decreased by PFOA treatment. Analysis of the cell cycle of thymocytes indicated that the thymic atrophy caused by PFOA in mice results, at least in part, from inhibition of thymocyte proliferation. Interestingly, in vitro exposure to PFOA for up to 24 h did not produce analogous effects in either thymocytes or splenocytes. Thus, the thymic and splenic atrophy caused by PFOA appears to involve an indirect pathway.

Involvement of the peroxisome proliferator-activated receptor alpha in the immunomodulation caused by peroxisome proliferators in mice.

Peroxisome proliferators (PPs) are a large class of structurally diverse chemicals, which includes drugs designed to improve the metabolic abnormalities linking hypertriglyceridemia to diabetes, hyperglycemia, insulin-resistance and atherosclerosis. We have recently demonstrated that exposure of rodents to potent PPs indirectly causes a number of immunomodulating effects, resulting in severe adaptive immunosuppression. Since the peroxisome proliferator-activated receptor alpha (PPARalpha) plays a central role in mediating the pleiotropic responses exerted by PPs, we have compared here the immunomodulating effects of the PPs perfluorooctanoic acid (PFOA) and Wy-14,643 in wild-type and PPARalpha-null mice. The reductions in spleen weight and in the number of splenocytes caused by PP treatment in wild-type mice was not observed in PPARalpha-null mice. Furthermore, the reductions in thymus weight and in the number of thymocytes were potently attenuated in the latter animals. Similarly, the dramatic decreases in the size of the CD4(+)CD8(+) population of cells in the thymus and in the number of thymocytes in the S and G2/M phases of the cell cycle observed in wild-type mice administered PPs were much less extensive in PPARalpha-null mice. Finally, in contrast to the case of wild-type animals, the response of splenocytes isolated from the spleen of PP-treated PPARalpha-null mice to appropriate T- or B-cell activators in vitro was not reduced. Altogether, these data indicate that PPARalpha plays a major role in the immunomodulation caused by PPs. The possible relevance of these changes to the alterations in plasma lipids also caused by PPs is discussed.