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The environmental pollutants perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) cause a dramatic reduction in the size of the major adipose tissue depots and a general body weight decrease when they are added to the food of mice. We demonstrate here that this is mainly due to a reduction in food intake; this reduction was not due to food aversion. Remarkably and unexpectedly, a large part of the effect of PFOA/PFOS on food intake was dependent on the presence of the uncoupling protein 1 (UCP1) in the mice. Correspondingly, PFOA/PFOS treatment induced recruitment of brown adipose tissue mitochondria: increased oxidative capacity and increased UCP1-mediated oxygen consumption (thermogenesis). In mice pair-fed to the food intake during PFOA/PFOS treatment in wildtype mice, brown-fat mitochondrial recruitment was also induced. We conclude that we have uncovered the existence of a regulatory component of food intake that is dependent upon brown adipose tissue thermogenic activity. The possible environmental consequences of this novel PFOA/PFOS effect (a possible decreased fitness) are noted, as well as the perspectives of this finding on the general understanding of control of food intake control and its possible extension to combatting obesity.

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.

We have previously demonstrated that severe adipose tissue atrophy occurs upon dietary treatment of mice with potent peroxisome proliferators (PPs). This atrophy occurs subsequent to peroxisome proliferation in the liver and may represent a novel addition to the pleiotropic effects exerted by PPs. In the present study we have characterized the recovery of mice from such atrophy following cessation of exposure. Following termination of treatment with perfluorooctanoic acid (PFOA) for 7 days, the adipose tissue atrophy was rapidly reversed, beginning on 2-5 days of recovery and being complete within 10 days. In contrast, hepatic peroxisome proliferation recovered much more slowly, indicating that these processes are not strictly coordinated. Analysis of lipoprotein lipase and hormone-sensitive lipase activities in adipose tissue revealed that the decrease and increase in these activities, respectively, caused by PFOA were both reversed within 10 days of recovery. Overall, these data provide further support for our previous conclusion that the adipose tissue atrophy induced by PFOA is caused, at least in part, by changes in the activities of lipoprotein lipase and hormone-sensitive lipase. The serum level of cholesterol, which increased after termination of PFOA treatment, returned to normal with a time-course similar to the recovery of adipose tissue weight, although hepatic peroxisome proliferation was still present. The possible relationship between the reduction in serum cholesterol and/or in its availability to peripheral tissues and the associated atrophy of adipose tissues caused by PPs is discussed.

Dimethyl sulfoxide (DMSO) is widely and routinely used as a vehicle in various investigations, especially within the pharmaceutical industry. It has been used for the evaluation of the effects of hydrophobic xenobiotics on cells, as well as for the cryopreservation of biological material. Isothermal microcalorimetry is a powerful tool for monitoring heat production, which is a function of general cellular metabolic activity. Employing this microcalorimetric technique, a low concentration of DMSO routinely used for the addition of hydrophobic substances to, e.g., cell cultures, was shown to decrease heat production (per unit DNA) by the rat hepatoma cell lines FAO, Morris 7800C1 and H4IIE by 32–38%. However, such low concentrations of DMSO did not influence the cell cycle or the degree of apoptosis in these cell populations. Caution is thus advisable when utilizing DMSO as a vehicle in cell culture experiments.

In previous studies in our laboratory (L. Mankowitz, L. Staffas, M. Bakke, and J. Lund, Biochem J, 1995, 305, 111–118; L. Staffas, L. Mankowitz, M. Soderstrom, A. Blanck, I. Porsch-Hallstrom, C. Sundberg, B. Mannervik, B. Olin, J. Rydstrom, and J.W. DePierre, Biochem J, 1992, 286, 65–72) isoenzymes of GST, primarily of the μ class, have been shown to be downregulated by adrenocorticotropic hormone (ACTH) in rat and mouse adrenal cells. In the present investigation the human adrenal H295R cell line (W.E. Rainey, I.M. Bird, and J.I. Mason, Mol Cell Endocrinol, 1994, 100, 45–50) was examined in a similar manner. Analysis by reverse-phase HPLC revealed that these cells express four isoenzymes of GST, i.e., A1, A2, P1, and M4, as well as another unidentified protein that was retained by our affinity column (elution time of 32 min) and, thus, presumably binds glutathione. Among these forms, A1 was present at the highest level. Upon addition of forskolin (an activator of adenylate cyclase which has been shown previously to mimic the effect of ACTH on adrenal cells) to the culture medium, the level of A1 decreased approximately 70% by forskolin, whereas the levels of the other isoenzymes were slightly increased, and that of the unknown form doubled. Thus, the influence of ACTH on expression of GST isoenzymes in this human adrenal cell line differs from that in rat and mouse adrenal cells. © 2002 Wiley Periodicals, Inc. J Biochem Mol Toxicol 16:169–173, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10034

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.

The regulation of the aldo-keto reductase AKR7A1 was examined in the livers of male and female rats during development by using Western blots, and its contribution to carbonyl metabolism was assessed by using enzyme assays. Hepatic levels of AKR7A1 are low in fetal rats and rise to a peak at around 6 weeks of age in animals of both sexes. Higher levels of the enzyme are found in adult male rat liver than in adult female rat liver. The reductase, therefore, appears to be subject to sex-specific regulation. The effect of growth hormone in mediating this difference in expression was examined by using hypophysectomized animals whose serum growth hormone levels had been feminized by continuous administration. Results demonstrate that such treatment leads to a reduction in AKR7A1 expression. AKR7A1 was found to be constitutively expressed in rat tissues such as liver, kidney, small intestine, and testis, but it was not detected in nasal mucosa, skeletal muscle, heart, adrenal gland, brain, or spleen. However, AKR7A1 was inducible by the synthetic antioxidant ethoxyquin in liver, kidney, and small intestine, but not in the other tissues examined. These results show that levels of this important detoxication enzyme vary considerably according to age and sex and that dietary antioxidants can also influence its level in several tissues.

The present study concerns conjugation of 1-naphthol in primary cultures of rat ovarian cells. Two phase II enzymes catalyzing conjugation, i.e. phenol sulfotransferase (P-SULT) and phenol UDP-glucuronosyltransferase (P-UGT), were measured using 1-naphthol as substrate. The rates of conjugation by the different cell types of the rat ovary were the same at low concentrations and short incubation times. However, after 20 h of incubation the rate of conjugation in cells isolated from ovaries enriched in corpora lutea (CL) exceeded the rate in cells isolated from ovaries enriched in preovulatory follicles. In addition, when the granulosa cells were removed from the preovulatory follicles, the rate of conjugation was 1.7-fold higher, i.e. in the theca/stroma cells. When the cells were incubated with 1-[14C]naphthol and conjugates were subsequently separated by thin-layer chromatography, naphthyl glucuronide was the only conjugate observed. Pentachlorophenol (PCP), a commonly used inhibitor of P-SULT, inhibited 1-naphthol conjugation 50% in cell cultures, as well as in microsomal preparations. alpha-Naphthoflavone (ANF) and ellipticine (ELP), both cytochrome P450 (CYP) inhibitors, affected the conjugation of 1-naphthol in different ways; ANF did not affect P-UGT activity in microsomal preparations, but inhibited 1-naphthol conjugation in cell cultures by as much as 90%. On the other hand, ELP inhibited the conjugation of 1-naphthol up to 99% in the cell cultures, but only 75% in microsomal fractions. Testosterone (TST) and estradiol inhibited this activity approximately equal 50% in both of these experimental systems. Clomiphene citrate (CLF), a drug used to induce ovulation and demonstrating both estrogenic and antiestrogenic effects, did not influence the conjugation of 1-naphthol significantly in the cell cultures. The present findings demonstrate that P-UGT is by far the major enzyme conjugating 1-naphthol in the rat ovary and that commonly used inhibitors of P-SULT and CYPs also inhibit P-UGT activity, either directly or via other mechanisms.

The levels and subcellular distribution of enzymes involved in defenses against reactive oxygen superoxide dismutase (SOD; E.C.1.15.1.1), glutathione peroxidase (GPX; E.C.1.11.1.9), catalase (CAT; E.C.1.11.1.6), and DT-diaphorase (DT; E.C.1.6.99.2) and of the conjugating enzymes glutathione transferase (GST; E.C.2.5.1.18) and p-sulphotransferase (p-ST; E.C.2.8.2.1) in the corpus luteum of ovaries from pregnant and non-pregnant pigs were investigated. In addition, non-protein thiols and glutathione reductase (GRD; E.C.1.6.4.2) were examined in the same manner. The total cytosolic activities of CAT, DT, GRD, and p-ST were significantly increased, whereas total GST activity was decreased in the pregnant corpus luteum compared to the corresponding activities in non-pregnant corpus luteum. In the case of the mitochondrial fraction from pregnant corpus luteum, GPX and GRD displayed significant increases in specific activity. Upon subfractionation of the mitochondrial fraction (i.e. mitoplast preparation), SOD activity was distributed equally between the mitoplasts and the supernatant. CAT and GPX activities were mainly recovered in the supernatant, while the major GRD activity pelleted with the mitoplasts. Microsomes from pregnant corpus luteum demonstrated increased specific GPX activity and decreased SOD activity compared to the non-pregnant corpus luteum. No differences in the non-protein thiol levels in the cytosolic, mitochondrial, or microsomal fractions from the corpus luteum were observed between non-pregnant and pregnant sows.

Some widely used antidepressants such as imipramine, clomipramine, and citalopram have been found to possess antineoplastic effects. In the present study, these compounds were found to induce apoptotic cell death in human acute myeloid leukemia HL-60 cells. Apoptosis induced by the antidepressants was identified by electron microscopy and conventional agarose gel electrophoresis and was quantitated by propodium iodide staining and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) via flow cytometry. Treatment with apoptosis-inducing concentrations of the antidepressants (80 microM imipramine, 35 microM clomipramine, or 220 microM citalopram) caused induction of caspase-3/caspase-3-like activity, which was monitored by the cleavage of poly(ADP-ribose) polymerase (PARP), the loss of the 32 kD caspase-3 (CPP32) precursor, and the cleavage of the fluorescent CPP32-like substrate PhiPhiLux. Pretreatment with a potent caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl-ketone (zVAD-fmk) inhibited antidepressant-induced CPP32/CPP32-like activity and apoptosis. Furthermore, activation of caspase induced by the antidepressants was preceded by the hypergeneration of intracellular reactive oxygen species (ROS). These results suggested that the antidepressants may induce apoptosis via a caspase-3-dependent pathway, and induction of apoptosis by the antidepressants may provide a clue for the mechanism of their antineoplastic effects.

The expression of different isoenzymes of glutathione transferase (GST), i.e. the cytosolic subunits GSTA1/A2, A3, A4, A5, M1/2, M2 and P1, T2, and the microsomal GST in follicles of different sizes and in corpora lutea from porcine ovary, was investigated by Western blotting. No immunoreactivity was obtained with anti-rat GSTT2 or anti-rat microsomal GST polyclonal antibodies. In contrast, GSTA1/A2, A3, A4, A5, M1/2, M2 and P1 are all expressed in the cytosol from porcine ovaries. In general, the highest levels of these GST isoenzymes were present in the cytosol from corpora lutea, in agreement with measurements of activity towards 1-chloro-2,4-dinitrobenzene. Imunoreactivity with anti-rat GSTP1 was only obtained with follicles. The cytosolic GSTs from follicles and corpora lutea were affinity purified on glutathione–Sepharose and separated by reversed-phase high-performance liquid chromatography in order to quantitate the different subunits. A peak corresponding to the class pi subunit was present in follicles. This peak was also seen with corpora lutea, although at very low level. There were four peaks containing class mu subunits. The remaining peaks were concluded to contain the class alpha subunits, except for two peaks which are suggested to contain proteins other than GSTs. The levels of the different subunits were quantitated on the basis of the areas under the peaks and the relative amounts in follicles of different sizes and in corpora lutea corresponded well with the Western blot analysis.

A novel and rapid procedure for the isolation of catalase from mouse liver, after prior treatment with the peroxisome proliferator perfluorooctanoic acid was developed using immobilized metal ion affinity chromatography involving chelation with zinc ions. The purification developed is simple, rapid (requiring 3 hours from cytosol or peroxisomal matrix to homogeneous proteins), reproducible, and yields virtually complete overall recovery of catalase activity. This procedure makes catalase from a variety of tissues and physiological and environmental conditions more readily available for study.

Human mono- and lymphocytes from peripheral blood and the monoblastoid cell line U-937 were used in this in vitro study of drug-induced lipidosis. Mono- and lymphocytes were exposed for 4 days to three different tricyclic antidepressants (TCAs), imipramine (25 microM), clomipramine (10 microM) and citalopram (80 microM). The lipophilic fluorophore Nile red, which stains intracellular lipid structures selectively, was used as a lipid probe. Fluorescence microscopy, spectrofluorimetry and flow cytometry were used to detect cellular lipidosis, as verified by electron microscopy. Our results demonstrate that imipramine, clomipramine and citalopram induce lipidosis in monocytes and U-937 cells, but not in lymphocytes. An accurate quantitation of induced intracellular lipidosis can be achieved by spectrofluorimetric and flow cytometric analysis.

The peroxisome proliferators perfluorooctanoic acid (PFOA; 0.02% w/w), perfluorodecanoic acid (PFDA; 0.02%, w/w), nafenopin (0.125%, w/w), clofibrate (0.5%, w/w), and acetylsalicylic acid (ASA; 1%, w/w) were administered to male C57 BL/6 mice in their diet for two weeks. Parameters for Fe3+ ADP, NADPH or ascorbic acid-initiated lipid peroxidation in vitro were measured. Approximately a twofold increase in susceptibility to lipid peroxidation was obtained for all the peroxisome proliferators tested. Cotreatment of mice with the peroxisome proliferator ASA (1%, w/w) and a catalase inhibitor, 3-amino-1,2,4-triazole (AT; 0.4%, w/w) for 7 days resulted in little inhibition of peroxisome proliferation, an elevated level of H2O2 in vivo, and total inhibition of the increased susceptibility to lipid peroxidation in vitro. No increase in lipid peroxidation in vivo was observed. Certain antioxidant enzymes (DT-diaphorase, superoxide dismutase, glutathione transferase, glutathione peroxidase, and glutathione reductase) and components (ubiquinone and alpha-tocopherol) were also measured. The results showed that there was some induction of these antioxidant enzymes and components by ASA or aminotriazole, except for glutathione peroxidase and superoxide dismutase, which were inhibited. The possible involvement of oxidative stress in the carcinogenicity of peroxisome proliferators is discussed.

Perfluorobutanesulfonyl fluoride (PBSF) has been introduced as a replacement for its eight-carbon homolog perfluorooctanesulfonyl fluoride (POSF) in the manufacturing of fluorochemicals. Fluorochemicals derived from PBSF may give rise to perfluorobutanesulfonic acid (PFBS) as a terminal degradation product. Although basic mammalian toxicokinetic data exist for PFBS, information on its tissue distribution has only been reported in one study focused on rat liver. Therefore, here we characterized the tissue distribution of PFBS in mice in the same manner as we earlier examined its eight-carbon homolog perfluorooctanesulfonate (PFOS) to allow direct comparisons. Following dietary exposure of adult male C57/BL6 mice for 1, 3 or 5d to 16 mg (35)S-PFBS kg(-1) d(-1), both scintillation counting and whole-body autoradiography (WBA) revealed the presence of PFBS in all of the 20 different tissues examined, demonstrating its ability to leave the bloodstream and enter tissues. After 5d of treatment the highest levels were detected in liver, gastrointestinal tract, blood, kidney, cartilage, whole bone, lungs and thyroid gland. WBA revealed relatively high levels of PFBS in male genital organs as well, with the exception of the testis. The tissue levels increased from 1 to 3 d of exposure but appeared thereafter to level-off in most cases. The estimated major body compartments were whole bone, liver, blood, skin and muscle. This exposure to PFBS resulted in 5-40-fold lower tissue levels than did similar exposure to PFOS, as well as in a different pattern of tissue distribution, including lower levels in liver and lungs relative to blood.

A novel protein that binds to a glutathione-Sepharose affinity column has been detected in mature, but not immature, rat ovary. This protein could be resolved from all identifiable components when the affinity-purified material, containing primarily glutathione transferases, was analyzed on reversed phase-HPLC. The unidentified protein migrated with an apparent molecular weight of 34 kDa on SDS-PAGE. After purification by affinity chromatography and subsequent preparative electrophoresis, the protein was subjected to N-terminal amino acid sequence analysis. The sequence obtained demonstrated a high degree of homology with an internal amino acid sequence in human carbonyl reductase (EC 1.1.1.184).

The membrane topology of rat liver microsomal glutathione transferase was investigated by comparing the tryptic cleavage products from intact and permeabilized microsomes. It was shown that lysine-4 of microsomal glutathione transferase is accessible at the luminal surface of the endoplasmic reticulum, whereas lysine-41 faces the cytosol. These positions are separated by a hydrophobic stretch of 25 amino acids (positions 11–35) which comprises the likely membrane-spanning region. Reaction of cysteine-49 of the microsomal glutathione transferase with the charged sulfhydryl reagent DTNB (2,2′-dithiobis(5-nitrobenzoic acid))) in intact microsomes further supports the cytosolic localization of this portion of the polypeptide chain. The role of two other potential membrane-spanning/associated segments in the C-terminal half of the polypeptide chain was examined by investigating the association of the protein to the membrane after trypsin cleavage at lysine-41. Activity measurements and Western blot analysis after washing with high concentrations of salt, as well as after phase separation in Triton X-114, indicate that this portion of the protein also binds to the membrane. It is also shown that cleavage of the purified protein at Lys-41 and subsequent separation of the fragments obtained yields a functional C-terminal polypeptide with the expected length for the product encompassing positions 42–154. The location of the active site of microsomal glutathione transferase was investigated using radiolabelled glutathione together with a second substrate. Since isolated rat liver microsomes do not take up glutathione or release the glutathione conjugate into the lumen, it can be concluded that the active site of rat liver microsomal glutathione transferase faces the cytosolic side of the endoplasmic reticulum.

The capacity of ovarian granulosa cells from rat and pig to release reactive metabolites produced from polycyclic aromatic hydrocarbons with the ability to cause mutations in neighbouring cells has been studied. For this purpose we have used cocultivation with V79 Chinese hamster cells as a detection system. The cells were treated with two different polycyclic aromatic hydrocarbons (PAHs), 7,12-dimethylbenz(a)anthracene (DMBA) or (-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene (BP-7,8-diol). The resulting mutation frequency in the V79 cells after cocultivation, as a function of granulosa cell number, DMBA, or BP-7,8-diol concentration and time, was determined. The progesterone concentration in the medium after cocultivation was also analyzed as a measure of the differentiation of the granulosa cells. These studies demonstrate an increasing frequency of mutations in the V79 cells with an increasing number of granulosa cells. The maximal number of mutations were achieved with a DMBA or BP-7,8-diol concentration of 5 or 2 microM, respectively. The optimal cocultivation time was 24 h. These results clearly show that the granulosa cells can bioactivate PAHs to reactive metabolites with the capacity to migrate into surrounding cells and cause mutations in these cells. Compounds metabolized to mutagenic products by granulosa cells might thus cause mutations in the neighbouring germ cells, with possible consequences for the offspring.

Male mice were exposed via their diet to perfluoro fatty acids of various chain-lengths (2-10 carbon atoms) at different doses (0.02 and 0.1% weight) and for different periods of time (2-10 days). Thereafter, we monitored effects on liver and body weights and a number of hepatic parameters, including mitochondrial protein content, microsomal contents of cytochromes P450 and b5, NADPH-cytochrome P450 reductase activity [measured as NADPH-cytochrome c reductase (EC 1.6.2.3)], microsomal and cytosolic epoxide hydrolase (EC 3.3.2.3) activities, cytosolic DT-diaphorase (EC 1.6.99.2), glutathione transferase (EC 2.5.1.18), glutathione peroxidase (EC 1.11.1.9) and superoxide dismutase (EC 1.15.1.1) activities, and levels of thiobarbituric acid-reactive material (as an indicator of lipid peroxidation) in the mitochondrial subfraction. The most dramatic changes observed were a 5-9-fold increase in mitochondrial protein, a 3-6-fold increase in the microsomal content of cytochrome P450, a 3-10-fold increase in cytosolic DT-diaphorase activity, an approximately 2-fold increase in cytosolic epoxide hydrolase activity and as much as a 60% decrease in the level of thiobarbituric acid-reactive compounds in the mitochondrial fraction. Smaller increases in microsomal epoxide hydrolase activity and decreases in cytosolic glutathione peroxidase activity were also observed. Of the perfluoro fatty acids tested, perfluorooctanoic acid caused the largest changes in the parameters examined here. Dietary exposure of mice to a 0.02% dose of this substance for 10 days results in a maximal or near-maximal effect in most cases.

Myelin bodies were isolated from the renal cortex of gentamicin-treated rats (100 mg/kg body weight, twice daily for 3 days, i.p.) employing an initial pelleting by differential centrifugation and subsequent flotation on a discontinuous sucrose gradient. These structures were found to contain almost twice as much protein as phospholipid and SDS-polyacrylamide gel electrophoresis revealed the presence of many different polypeptides. All the major phospholipids are present, although myelin bodies contain a considerably higher proportion of phosphatidylinositol, somewhat more phosphatidylcholine and considerably lower percentages of phosphatidylserine and sphingomyelin than do normal renal phospholipids. The fatty acids of myelin body phospholipids are highly saturated (67.3-87.9%) and a striking feature is the occurrence of relatively large amounts of 22:1, presumably erucic acid, especially in sphingomyelin. Myelin bodies contain small amounts of unesterified cholesterol, unesterified dolichol and coenzymes Q9 and Q10.

After hypophysectomy, the level of glutathione transferase subunit 4 increases in the adrenal, as well as in the liver, as do those of several other forms of glutathione transferase. This increase in subunit 4 can subsequently be down-regulated by administration of adrenocorticotropin. The present investigation demonstrates that also in primary cultures of female rat adrenal cells an increase in the level of glutathione transferase subunit 4 (as shown by immunoblotting) occurs in the absence of adrenocorticotropin. When adrenocorticotropin or dibutyryladenosine 3',5'-phosphate was administered to these cells, a down-regulation of this enzyme level was observed, in agreement with the in vivo situation. This down-regulation was not affected by aminoglutethimide, an inhibitor of the cholesterol-side-chain-cleavage enzyme (cytochrome P-450scc) which is the rate-limiting step in the biosynthesis of steroids. Hence adrenal steroid production is not involved in the down-regulation of glutathione transferase subunit 4 by adrenocorticotropin.

Male C57bl/6 mice were administered clofibrate (0.5%, w/w), nafenopin (0.12%, w/w) or WY-14.643 (0.125%, w/w) in their diet for 4 days. Assay of eight mitochondrial marker enzymes, - i.e., malate and glutamate dehydrogenases (matrix markers), cytochrome oxidase and cytochromes c + c1 and a (inner membrane), adenylate kinase (intermembrane space) and monoamine oxidase and microsomal glutathione transferase (outer membrane) - and morphometric analysis of electron micrographs was used to examine hepatic mitochondria after treatment with these peroxisome proliferators. A moderate increase in the number of hepatic mitochondrial profiles, with a simultaneous decrease in the average size of these organelles, was observed. The total mitochondrial volume is apparently unchanged during this process. An important experimental consequence of the apparent decrease in mitochondrial size is the redistribution of a large portion of the total hepatic mitochondria from the ‘nuclear’ to the mitochondrial fraction. A similar effect was seen with rats.

The present study demonstrates that during the first 1.5-3 min after a single intraperitoneal administration of [3H]gentamicin to rats, most of the radioactivity in the kidney cortex is recovered in the cytosolic and microsomal fractions upon subcellular fractionation. Subsequently, the level of radioactivity recovered in the cytosolic fraction decreases markedly, whereas this level remains relatively unchanged in microsomes and increases somewhat in the nuclear and mitochondrial fractions. A steady state is apparently reached 13 hr after the injection. The high initial concentration of gentamicin in the cytosol may indicate that this substance is taken up to a large extent by diffusion. Such uptake is somewhat surprising, because of the polar nature of gentamicin. The small size of this drug may, however, allow it to diffuse through so-called pores and/or interaction with negatively charged phospholipids may be involved in the uptake of gentamicin. The initial total level of radioactivity recovered in microsomes after in vivo administration of [3H]gentamicin was considerably higher than in the nuclear and mitochondrial-lysosomal fractions. Furthermore, when gentamicin was added directly to kidney homogenate prepared from untreated rats, instead of being administered in vivo, this substance was still recovered in highest amounts in the total microsomal fraction. This observation may indicate that enrichment of gentamicin in the endoplasmic reticulum (or fragments thereof) reflects a special affinity of this drug for these membranes and is probably not the result of a particular in vivo process. There was no difference in the levels of radioactivity recovered in smooth and rough microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of hypophysectomy and subsequent treatment with adrenocorticotropic hormone (adrenocorticotropin, ACTH) on the isoenzymes of glutathione transferase in the rat adrenal gland was investigated. A large increase (approx. 11-fold) in the level of transferase subunit 4 was observed in hypophysectomized animals by immunoblotting. When the activity of glutathione transferase 4-4 was measured in adrenal cytosol using trans-stilbene oxide as a selective substrate, a 15-fold increase was noted. Lack of the pituitary hormone ACTH is apparently related to this increase, since treatment of hypophysectomized animals with ACTH for 2 weeks partially down-regulated subunit 4. Glutathione transferase subunits 3 and 8 in the adrenal were also increased in amount by hypophysectomy, but not at all to the same extent. The activity of glutathione transferase 4-4 was elevated also in the liver and ovary (5 and 1.5 times respectively) after hypophysectomy. These elevated enzyme levels were, however, not affected by ACTH treatment. This down-regulation of glutathione transferases in the rat adrenal by ACTH may be related to the fact that, under normal conditions, this organ is highly susceptible to the toxic effects of various polycyclic hydrocarbons, whereas under circumstances where there is no ACTH production, as in hypophysectomized rats, the adrenal is resistant to these same hydrocarbons.

A detailed subfractionation of the non-pregnant porcine corpus luteum (CL) was performed employing differential centrifugation. Marker enzyme assays (i.e., lactate dehydrogenase for the cytosol, NADPH-cytochrome P450 reductase for the endoplasmatic reticulum, catalase (CAT) for peroxisomes, glutamate dehydrogenase for the mitochondrial matrix and acid phosphatase for lysosomes) in all subfractions obtained exhibited a pattern of distribution similar to that observed with rat liver. These subfractions should be useful in connection with many types of future studies. In disagreement with previous biochemical and morphological studies, peroxisomes (identified on the basis of catalase activity and by Western blotting of catalase and of the major peroxisomal membrane protein (PMP-70)) sedimented together with mitochondria (i.e., at 5000×gav for 10 min) and not in the post-mitochodrial fraction prepared at 30,000×gav for 20 min by Peterson and Stevensson [Biochim. Biophys. Acta 1135 (1992) 207]. No other classical peroxisomal enzymes were detectable in the porcine ovary, raising questions concerning the function of peroxisomes in this organ. Furthermore, UDP-glucuronosyltransferase (UGT), generally considered to be an integral membrane protein anchored in the endoplasmatic reticulum, was recovered in both the cytosolic (i.e., the supernatant after centrifugation at 50,000×gav for 1 h) and the microsomal fraction of the porcine corpus luteum, even upon further centrifugation of the former. In contrast, UGT sediments exclusively in the microsomal fraction upon subfractionation of the liver and ovary from rat.

Acyl-CoA thioesterases hydrolyze acyl-CoAs to free fatty acids and CoASH, thereby regulating fatty acid metabolism. This activity is catalyzed by numerous structurally related and unrelated enzymes, of which several acyl-CoA thioesterases have been shown to be regulated via the peroxisome proliferator-activated receptor alpha, strongly linking them to fatty acid metabolism. Two protein families have recently been characterized, the type I acyl-CoA thioesterase gene family and the type II protein family, which are expressed in cytosol, mitochondria and peroxisomes. Still, only little is known about regulation of their expression and precise functions in vivo. In the present study, we have investigated the activity and expression of acyl-CoA thioesterase in the porcine ovary during different phases of the estrus cycle. The activity was low in homogenates obtained during the immature and follicular phases, increasing nearly 4-fold during the luteal phase, with the highest activity being found in the pregnant corpus luteum (about 7-fold higher than in immature follicles). The increase in homogenate activity in corpus luteum from pregnant pigs was due to a moderate increase in the cytosolic activity, and an approximately 20-25-fold increase in the mitochondrial fraction. Western blot analysis showed no detectable expression of the type I acyl-CoA thioesterases (CTE-I and MTE-I) and revealed that the increased activity in cytosol and mitochondria is due to increased expression of the type II acyl-CoA thioesterases (CTE-II and MTE-II). This apparent hormonal regulation of expression of the type II acyl-CoA thioesterase may provide new insights into the functions of these enzymes in the mammalian ovary.

In the present study, we characterized the effects of peroxisome proliferators (PP) on adipose tissue in mice. Treatment with potent PP, such as perfluorooctanoic acid (PFOA), 2-methyl-2-(p(1,2,3,4-tetrahydroxy-naphthyl)-phenoxy)propionic acid, (4-chloro-6-(2,3-xylidino)2-pyrimidinylthio) acetic acid, and di(2-ethylhexyl)phthalate, caused dramatic decreases in adipose tissue weight, whereas the moderately potent PP, acetylsalicylic acid, had a relatively weak effect. This decrease in weight reflects a loss of fat from adipocytes rather than a loss of cells, as demonstrated by constant DNA content. The dose-dependency and time-course experiments indicate that peroxisome proliferation occurs simultaneously with or prior to adipose tissue atrophy. Thus, hepatic peroxisome proliferation might result in the increased mobilization of lipids and lipid utilization in liver. The enhanced adipose tissue hormone-sensitive lipase (HSL) activity and down-regulated lipoprotein lipase (LPL) activity observed upon PP treatment might, at least in part, explain the loss of fat via increased FA release from adipocytes and/or decreased FA uptake from the circulation, respectively. In addition, the possible involvement of the increased tumor necrosis factor alpha expression found upon PFOA treatment in reducing the insulin sensitivity of adipose tissue and thereby altering LPL and HSL activities is discussed.

In earlier experiments, adrenocorticotropic hormone (ACTH) was shown to decrease the level of glutathione transferase M1 (murine glutathione transferase mu-1) (mGSTM1), as well as of the corresponding mRNA, in a murine adrenocortical cell line. In the present study, the effect of ACTH on mGSTM1 gene transcription was examined using two techniques. First, a cDNA that coded for the mGSTM1 subunit but lacked the corresponding promoter sequences was transfected into the adrenocortical cell line, and the effect of ACTH on the level of the corresponding transcript was compared to that of endogenous mGSTM1 mRNA. The other technique used was nuclear run-on transcription, where the rate of transcription of endogenous mGSTM1 mRNA in ACTH-treated cells was compared to that in untreated control cells. These experimental approaches indicated that the rate of transcription of the mGSTM1 gene is regulated by ACTH in adrenocortical cells. © 2001 John Wiley & Sons, Inc. J Biochem Mol Toxicol 15:128–132, 2001

In order to investigate the molecular mechanism of the antineoplastic effects exerted by the antidepressive agents imipramine, clomipramine, and citalopram, we examined the effects of these compounds on cell viability, generation of reactive oxygen species (ROS), and mitochondrial membrane potential (DeltaPsi(m)) in human acute myeloid leukemia HL-60 cells. Our results indicate that exposure to these compounds causes a loss in cell viability by activating the apoptotic process, as identified by electron microscopy, DNA gel electrophoresis, and flow cytometry. The increased generation of ROS induced by these drugs was a relatively early event and preceded the loss of DeltaPsi(m). Overexpression of the antiapoptotic protein Bcl-2 or Bcl-X(L) prevents antidepressant-induced apoptosis, as well as loss of DeltaPsi(m), but does not affect the generation of ROS.

Cationic amphiphilic drugs, in general, induce phospholipid disturbances. Tricyclic, as well as other antidepressants belong to this group. In experimental animals, antidepressants induce lipid storage disorders in cells of most organs, a so-called generalized phospholipidosis. This disorder is conveniently detected by electron microscopic examination revealing myelin figures. Myelin figures or myeloid bodies are subcellular organelles containing unicentric lamellar layers. The lipidotic induction potency during in vivo is related to the apolarity of the compound. Metabolism of phospholipids takes place within the cell continuously. Several underlying mechanisms may be responsible for the induction of the phospholipid disturbance. For instance, it has been suggested that the compounds bind to phospholipids and such binding may alter the phospholipid’s suitability as a substrate for phospholipases. Free TCA or metabolites thereof may also inhibit phospholipases directly, as has been demonstrated for sphingomyelinase in glioma and neuroblastoma cells. Both these mechanisms might result in phospholipidosis. Interaction between drug and phospholipid bilayer has been investigated by nuclear magnetic resonance technique. There seems to be large differences in the sensitivities amongst different organs. Steroid-producing cells of the adrenal cortex, testis and ovaries are in particular susceptible to drug-induced lipidosis. The so-called foam cells are lung macrophages located in the interstitium which become densely packed with myelin figures during TCA exposure. It requires about 3–6 weeks of treatment to develop this converted cell. In cell cultures however, phospholipidosis is demonstrated already after 24 h only. It appears that the cells that undergo TCA-induced lipidosis may recover after withdrawal of the drug. The time required to achieve complete recovery ranges from 3–4 weeks to several months, depending on the organ affected. Little is known about the functional significance of lipidosis. Even if TCA and other antidepressants show other effects, it has not been possible to exclusively relate it to phospholipidosis. However, few attempts have been made to correlate the physiological effects of TCAs in experimental animals to the morphological changes associated with phospholipidosis. There is an increasing evidence however, that cationic amphiphilic drugs may have effects on immune function, signal transduction and receptor-mediated events, effects that to some extent might be related to disturbances in phospholipid metabolism.

Male and female C57B1/6 mice were rendered vitamin A-deficient, and the effects of this deficiency on certain xenobiotic-metabolizing enzymes and defenses against oxidative stress were examined. Vitamin A deficiency significantly increased the levels of DT-diaphorase, glutathione transferase, and catalase in the hepatic cytosolic fraction from male mice (5.2-, 1.6-, and 3.5-fold, respectively), as well as from female mice (4.8-, 3.3-, and 2.4-fold, respectively). In the hepatic mitochondrial fraction (containing peroxisomes) from male animals, the activities of urate oxidase and catalase were increased 3.4- and 1.7-fold, respectively. The activity of catalase in the mitochondrial fraction from female mice was not affected by vitamin A deficiency, whereas the activity of peroxisomal urate oxidase was increased 2.9-fold. The hepatic level of ubiquinone was increased somewhat. The significance of the increases observed here is presently unclear, but it may be speculated that vitamin A and/or its metabolites are somehow involved in the down-regulation of these proteins. Another possibility is that these enzymes are increased as a result of hepatic oxidative stress caused by vitamin A deficiency. However, vitamin A deficiency had no effect on the activity of superoxide dismutase in this study, whereas the activity of glutathione peroxidase was slightly decreased (27%) in the hepatic cytosolic fraction from male mice. In addition, the hepatic level of alpha-tocopherol was decreased dramatically in the vitamin A-deficient animals.

The effects of perfluorooctanoic acid (PFOA), a potent hepatocarcinogen and peroxisome proliferator in rodents, on human cells have not yet been examined. In the present study we demonstrate that treatment of human hepatoblastoma HepG2 cells with PFOA induces apoptosis, as well as perturbs the cell cycle. This apoptosis was characterized by electron microscopy, which revealed typical nucleosomal fragmentation (also observed as a 'DNA ladder' upon electrophoresis on agarose) and was quantitated using propidium iodide staining of cellular DNA and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. This process was dose- and time-dependent: apoptosis became manifest with 200 microM and maximal (45% of the cells) upon exposure to 450 microM PFOA for 24 h. Electrophoresis of the DNA from HepG2 cells exposed to 500 microM PFOA for 24 h or to 400 microM PFOA for 48 h revealed a smear typical of non-specific degradation. These findings indicate that in the presence of high concentrations of PFOA for long times, HepG2 cells undergo primary and secondary necrosis. Quantitation of trypan blue exclusion supported this conclusion. Flow cytometric analysis revealed that the cell cycle of HepG2 cells was perturbed by exposure to 50-150 microM PFOA. A 50 microM concentration resulted in a significant increase in the proportion of G(2)/M cells and, simultaneously, a decrease in the number of cells in the S phase, whereas treatment with 100 or 150 microM PFOA increased the proportion of cells in the G(0)/G(1) phase and decreased the number of cells in the G(2)/M and S phases. Simultaneous flow cytometric analysis of apoptosis-associated DNA strand breaks using the TUNEL procedure and of propidium iodide staining of cellular DNA revealed DNA breaks in HepG2 cells exposed to 150 microM PFOA, prior to nuclear fragmentation.

The class Alpha glutathione S-transferase (GST) subunit A5 is expressed in the livers of young male and female rats. After sexual maturation, this protein is no longer detectable in the livers of male rats, but is still expressed in female rats. We have previously demonstrated that the sexually dimorphic secretion of growth hormone regulates the levels of certain class Mu GSTs in rat liver, and this study was designed to investigate the hormonal regulation of GSTA5. Control and hypophysectomized rats of both sexes were used to study the role of growth hormone in the regulation of hepatic GSTA5; and the influence of testosterone on the expression of this same subunit was investigated in intact females and castrated males. Liver cytosols were subjected to SDS/PAGE and immunoblotting using antibodies directed towards rat (r)GSTA5, and to affinity purification on glutathione–Sepharose followed by reverse-phase HPLC in order to quantify the relative levels of rGSTA1, A2, A3, A4, M1 and M2 subunits. These analyses revealed that the expression of rGSTA5 is, indeed, regulated by both growth hormone and testosterone.

We have recently reported that the tricyclic antidepressants (TCAs) imipramine, clomipramine, and citalopram induce apoptosis in human peripheral lymphocytes. This system is well suited for studies on the pathophysiology/physiology of apoptosis regulation. Apoptosis was determined using both DNA gel electrophoresis and flow cytometric analysis. TCA-induced apoptosis in lymphocytes was monitored in the presence of the protein synthesis inhibitor cycloheximide (CHX), the RNA synthesis inhibitor actinomycin D (Act D), the antioxidant reduced glutathione (GSH), the nuclease inhibitor aurintricarboxylic acid (ATA), the cytokine interleukin-2 (IL-2), and the immunostimulator linomide. CHX and Act D failed to prevent and actually enhanced TCA-induced apoptosis in lymphocytes, indicating that protein and RNA syntheses are not required for this process. Exogenous IL-2, GSH, and ATA protected the lymphocytes from apoptosis induced by TCAs in a dose-dependent manner, whereas linomide had no effect on TCA-induced apoptosis under our in vitro conditions. Our data demonstrate that TCA-induced apoptosis in human lymphocytes shares many common features with other stimuli-induced apoptotic processes.

By employing microcalorimetry to assess overall metabolic activity in combination with other assays for specific metabolic events, we have investigated the influence of cyclosporin A and FK506 on the metabolic status of resting and mitogen-stimulated human peripheral lymphocytes. Both cyclosporin A and FK506 significantly reduced heat output from resting lymphocytes. This reduction could not be correlated with effects on DNA synthesis, lactate production, ATP levels or mitochondrial uptake of Rhodamine 123. These two drugs also potently reduced the increase in heat output seen during mitogen stimulation of lymphocytes. Both cyclosporin A and FK506 also prevented the increase in DNA synthesis, lactate production and ATP levels seen in response to mitogen stimulation. The increase in mitochondrial uptake of Rhodamine 123 during blastoid transformation was significantly reduced only by cyclosporin A. We ascribe the major part of the effects of these compounds to inhibition of the glycolytic pathway in both resting and mitogen-stimulated lymphocytes. These results indicate that the immunosuppressants cyclosporin A and FK506 exert other effects on lymphocytes than their well-established inhibitory action on calcineurin.© 1997 Elsevier Science B.V. All rights reserved.

Vitamin A-adequate and vitamin A-deficient C57B1/6 mice were treated for ten days with 0.02% (w/w) perfluorooctanoic acid (PFOA) in their diet. Treated vitamin A-adequate and -deficient mice demonstrated approximately the same increases in liver somatic index (g liver/g body weight) (somewhat more than 2-fold) and mitochondrial protein content (5-fold). PFOA treatment resulted in a 26-fold increase in hepatic peroxisomal lauroyl-CoA oxidase activity in vitamin A-adequate mice, whereas the same activity was unchanged in vitamin A-deficient mice. Vitamin A deficiency itself caused a 3- to 4-fold increase in cytosolic catalase activity and a smaller increase in the activity of microsomal cytochrome P-450 IVA (lauric acid ω- and ω-1 hydroxylase) in this same organ. The induction of the activities of these enzymes was less prominent in vitamin A-deficient mice compared with the effect caused by PFOA in vitamin A-adequate mice, resulting in approximately the same maximal values for these parameters in both groups (i.e. approx. 21 mmol/g liver · min and 350 nmol/g liver · min, respectively). A 70 kDa protein, presumably the multifunctional protein, was shown by Commassie blue staining of SDS-polyacrylamide gels and by immunoblotting (with antibodies towards the multifunctional protein) to be induced to approximately the same degree in vitamin A-adequate and -deficient mice. A morphometric study revealed that PFOA causes the same extent of hepatic peroxisome proliferation in vitamin A-deficient as in vitamin A-adequate mice. The possibility that PFOA exerts its effect in vivo through at least two different mechanisms is discussed.

The effects of three peroxisome proliferators on the mRNA levels for some mitochondrial inner-membrane proteins in rat liver were investigated. Clofibrate, perfluorooctanoic acid, and acetylsalicylic acid all increased the mRNA levels for the mitochondrial-encoded respiratory-chain components cytochrome c oxidase subunit I and NADH dehydrogenase subunit I. Mitochondrial 16S rRNA was also induced by clofibrate. The mRNA levels for the nuclear-encoded mitochondrial inner-membrane proteins adenine nucleotide translocator and cytochrome c 1 were selectively induced by the different peroxisome proliferators. Malic enzyme, which is induced by thyroid hormone, was also induced by the three peroxisome proliferators tested. These effects are in some ways similar to those obtained with thyroid hormone.

Morris hepatoma 7800C1 cells (a Wistar rat cell line) were exposed to 100 microM arachidonic acid in the medium for seven days. This treatment resulted in 150% and 60% increases (above control activities) in acyl-CoA oxidase (which catalyzes the first step in peroxisomal beta-oxidation) and catalase activities, respectively. Arachidonic acid (C20:4) can be metabolized to 20- and 19-hydroxy-arachidonic acid by cytochrome P-450IVA and it was shown that our cells are capable of forming 20-hydroxyarachidonic acid. However, 20-hydroxyarachidonic acid (0.1-0.8 microM, 4 days) had no effects on lauroyl-CoA oxidase and catalase activities in Morris hepatoma cells. Treatment of 7800C1 cells with 100 microM all-trans-retinoic acid resulted in inductions of catalase (160% above the control activity) and carnitine acetyltransferase (140% above the control activity) activities. The activity of lauroyl-CoA oxidase was often, but not always, slightly induced by treatment with all-trans-retinoic acid. When all-trans-retinoic acid was administered together with arachidonic acid, these two compounds had a synergistic effect on the induction of acyl-CoA oxidase activity (almost 700% above the control activity). However, treatment of Morris hepatoma cells with the man-made peroxisome proliferator, perfluorooctanoic acid, together with all-trans-retinoic acid did not result in any synergistic effect on this same enzyme activity. In summary, this study (1) corroborates findings from transfection experiments indicating that the heterodimer PPAR-RXR alpha activates transcription of the acyl-CoA oxidase gene using the Morris hepatoma cell line; (2) shows that arachidonic acid induces the activity of lauroyl-CoA oxidase; (3) suggests that transcription of the catalase gene is not regulated by a PPAR-RXR alpha heterodimer in this system; and (4) demonstrates that peroxisome proliferation in Morris hepatoma cells by perfluorooctanoic acid is not as dependent on the level of retinoic acid as is the same process caused by arachidonic acid.

In this study, Morris hepatoma 7800C1 cells (from rat) were exposed to 500 microM perfluorooctanoic acid (PFOA) in the culture medium for 7 days. This treatment resulted in inductions of catalase, lauroyl-CoA oxidase (which catalyzes the first step in peroxisomal beta-oxidation) and of cytochrome P-450IVA (specialized for omega- and omega-1 hydroxylation of fatty acids). Northern blot analysis revealed that the level of mRNA for peroxisomal fatty acyl-CoA oxidase was enhanced in cells treated with PFOA. Inductions of the enzymes mentioned above are generally connected with peroxisome proliferation in vivo. This work also includes a comparison between the activities of catalase, lauroyl-CoA oxidase, DT-diaphorase and glutathione transferase in rat liver homogenate and 7800C1 cells in order to investigate to what extent this cell line differs from the situation in vivo. The findings suggest that the cells selectively lost most of their peroxisomes during transformation into a cell line and subsequent propagation. The control activities of catalase and lauroyl-CoA oxidase (marker enzymes for peroxisomes) were only about 2% of the corresponding enzyme activities in rat liver. In addition, a morphological study revealed that the frequency of peroxisomes in 7800C1 cells is very low. The control activity of glutathione transferase in 7800C1 cells was 11% of the corresponding activity in rat liver homogenate, whereas the level of DT-diaphorase was virtually the same in 7800C1 cells as in rat liver. Electron microscopic investigation of the control cultures revealed all signs of viable cells, with well-developed cell organelles. Treatment of 7800C1 cells with 500 microM PFOA has little effect on cellular morphology.

Male C57 BL/6 mice were exposed to 1.0% (w/w) acetylsalicylic acid (ASA) in their diet for 10 days and effects related to peroxisome proliferation were subsequently examined. A 2.2-fold increase in mitochondrial protein content was obtained. The activities of the peroxisomal enzymes, lauroyl-CoA oxidase, palmitoyl-CoA oxidation and catalase, were enhanced 4.5-, 4.0- and 2.1-fold, respectively. There was a dramatic increase (9.1-fold) in microsomal cytochrome P450 IVA-catalysed activity, a 1.6-fold induction of total microsomal P450 content and a 2-fold induction of microsomal cytochrome P450 reductase activity (measured as NADPH-cytochrome c reductase). Catalase activity in the cytosol was induced 5.2-fold and DT-diaphorase activity was increased 3.5- and 3.2-fold in the cytosol and mitochondria, respectively. There was a significant increase in the susceptibility of microsomes to lipid peroxidation. Smaller increases in superoxide dismutase, glutathione transferase and glutathione peroxidase activities were also observed. The possible relevance of these effects to the pharmacology of ASA is discussed.

Microsomal epoxide hydrolase from the human adrenal gland was purified to a high degree of homogeneity in 10% overall yield using sequential chromatography on DE-52, FPLC Mono Q and FPLC Superose columns. The fact that the overall purification was only 7.3-fold indicates that approx. 14% of the total microsomal protein consisted of this enzyme, a uniquely high value. The human adrenal enzyme was found to resemble rat liver microsomal epoxide hydrolase closely in a number of respects, including molecular weight, N-terminal amino-acid sequence and response to low-molecular weight ligands. However, rabbit antibodies directed against human adrenal microsomal epoxide hydrolase crossreacted only weakly with the corresponding rat liver protein. The relatively high levels of microsomal epoxide hydrolase in the human adrenal gland suggest that this enzyme may be of particular importance in this tissue. However, very little cytochrome P-450-catalyzed metabolism of xenobiotics has been demonstrated in the human adrenal and our present results speak against the involvement of microsomal epoxide hydrolase in the steroid metabolism of this gland. Thus, the function of this enzyme in the human adrenal is enigmatic.

Male C57BL/6 mice were exposed to 1% (w/w) (+)- or (-)-2-ethylhexanoic acid or an equimolar mixture of these enantiomers in their diet for 4 or 10 days. A significant increase in liver weight and a 2- to 3-fold increase in the protein content of the mitochondrial fraction were seen in all cases. Peroxisomal palmitoyl-CoA oxidation was increased 2- to 3.5-fold after 4 days of treatment and 4- to 5-fold after 10 days, while the corresponding increases in peroxisomal lauroyl-CoA oxidase activity were 2- to 3-fold and 9- to 12-fold, respectively. Peroxisomal catalase activity was unchanged, whereas the microsomal and cytosolic activities were increased 2- to 3-fold and 6- to 16-fold, respectively. These treatments also induced microsomal omega-hydroxylation of lauric acid 7-fold and soluble epoxide hydrolase activity in the mitochondrial and cytosolic fractions, as well as microsomal epoxide hydrolase activity about 50-100%. The only significant differences observed between the effects of (+)-2-ethylhexanoic acid and its (-)-enantiomer were on peroxisomal palmitoyl-CoA oxidation and lauroyl-CoA oxidase activity after 4 days of treatment. In both these cases the (+)-enantiomer resulted in increases which were 50-75% greater than those seen with the (-)-form.

Perfluorooctane sulfonic acid is almost as potent as perfluorooctanoic acid in causing increases in peroxisomal fatty acid beta-oxidation, peroxisomal catalase activity, omega-hydroxylation of lauric acid, cytosolic epoxide hydrolase activity and cytosolic DT-diaphorase activity. Octane sulfonic acid was ineffective at doses used for perfluorooctane sulfonic acid and perfluorooctanoic acid. The results support the theory of co-regulation of these parameters and peroxisome proliferation. The fact that perfluorooctane sulfonic acid causes peroxisome proliferation challenges the hypothesis that the first step in this process is formation of a thioester between the proliferator (the carboxylic group) and coenzyme A.

Dietary treatment of male C57B1/6 mice with clolibrate, nafenopin or WY-14.643 resulted in a modest (at most 2-fold) increase in the total catalase activity in the whole homogenate and mitochondrial fraction prepared from the livers of these animals. On the other hand, the catalase activity recovered in the cytosolic fraction was increased 12- to 18-fold, i.e. 30–35% of the total catalase activity in the hepatic homogenate was present in the high-speed supernatant fraction after treatment with these peroxisome proliferators. A study of the time course of the changes in peroxisomal and cytosolic catalase activities demonstrated that the peroxisomal activity both increased upon initiation of exposure and decreased after termination of treatment several days after the increase and decrease, respectively, in the corresponding cytosolic activity. This finding suggests that the cytosolic catalase may be on its way to incorporation into peroxisomes.

Immunoblot experiments and reverse-phase h.p.l.c. were used to study the levels of glutathione transferase subunits 1, 2, 3, 4, 6, 7 and 8 in the liver and adrenal of intact and hypophysectomized male and female Sprague-Dawley rats. A sexual dimorphism in the levels of several of these isoenzymes and in their responses to hypophysectomy was demonstrated. In the liver of sham-operated females and males there are differences in glutathione transferase activities and isoenzyme pattern. H.p.l.c. analysis showed higher levels of subunits 1, 3 and 4 in male rats compared with females. In contrast with the pronounced sex differences in sham-operated rats, the isoenzyme patterns of hypophysectomized males and females were very similar. In the adrenal glands, however, a sexual dimorphism became apparent only after hypophysectomy, when the level of subunit 4 was increased 14-fold in the female, whereas the corresponding increase in the male rat was only 2.7-fold. The hepatic pattern of glutathione transferase subunits could be altered by continuous infusion of growth hormone to both sham-operated and hypophysectomized rats of both sexes. This treatment feminized the isoenzyme pattern in sham-operated males and a similar effect was obtained upon treating hypophysectomized rats with thyroxine, cortisone acetate and a continuous infusion of growth hormone.

Male and female C57Bl/6 mice were administered perfluor-octanoic acid PFOA; 0.02-0.05% w/w; 5-10 days) in their diet. This treatment resulted in a several-fold induction of hepatic peroxisomal fatty acid beta-oxidation (monitored as increases in cyanide-insensitive palmitoyl-CoA oxidation, lauroyl-CoA oxidase and catalase activity) in all animals. The protein content of the hepatic mitochondrial fraction was also increased in all mice exposed to PFOA. Furthermore, studies on xenobiotic-metabolizing enzymes revealed no sex-related difference in the response to PFOA. All mice demonstrated a dramatic increase in omega-hydroxylation of lauric acid. Cytosolic epoxide hydrolase, glutathione transferase and DT-diaphorase activities were increased about 2-5-fold. These results with mice differ dramatically from previous studies and our own experiments here with Wistar rats, in which exposure to PFOA causes hepatic peroxisome proliferation in male animals, whereas females are unaffected.

C57B1/6 male mice were exposed through their diet to 11 dicarboxylic acids, carboxylic acids and diethyldicarboxylesters for 10 days. For the diacids and diethylesters this treatment resulted in a chain length-dependent induction of lauryl-CoA oxidase and cyanide-insensitive palmitoyl-CoA oxidation activities. A chain length of 12 carbon atoms or more seemed to be necessary for induction of these two activities. In addition, the same chain length dependence was observed for induction of lauric acid ω + ω-1 hydroxylase activity and increase in the protein content of the mitochondrial fraction. Treatment with two “natural” fatty acids, i.e. lauric and palmitic acid gave no effect at all on these various parameters. In no case was induction of cytosolic and mitochondrial epoxide hydrolase activities observed. Instead, a slight decrease in these activities was observed after administration of diacids with a chain length of 4–8 carbon atoms, whereas microsomal epoxide hydrolase activity was concurrently induced.

The distribution of glutathione transferase subunits 1, 2, 3, 4, 7 and 8 in the different cells of the female and male rat adrenal and the effects of hypophysectomy on these isoenzymes were studied using immunohistochemical methods. All these glutathione transferase subunits, with the exception of subunit 1, were present in the adrenal. Each subunit showed, however, its own characteristic distribution pattern. After hypophysectomy, increased staining for these isoenzymes was generally observed, and this effect was also cell-specific. Staining for subunit 2 increased in intensity in the zona fasciculata and reticularis after hypophysectomy, whereas a decrease was observed in the zona glomerulosa. Staining for subunit 8 was increased in the borderline between the capsule and zona glomerulosa, as well as in medullary chromaffin cells after hypophysectomy. The Mu subunits 3 and 4 increased markedly in fascicular and reticular cells after hypophysectomy and staining for subunit 3 was also increased in the medullary cells. A slight, but more general, increase was observed for subunit 7. We conclude from these experiments that the increases in glutathione transferase subunits observed in the rat adrenal after hypophysectomy are due to increased protein synthesis and/or increased protein stability and not to a selective destruction of cells lacking, or with low levels of, the isoenzymes.

Digitonin permeabilization of hepatocytes from control and clofibrate-treated (0.5% by mass, 10 days) male C57bl/6 mice was used to study the intracellular distributions of soluble (‘cytosolic’) epoxide hydrolase and of catalase. The following conclusions were drawn. (1) About 60% of the total soluble epoxide hydrolase activity in control mouse hepatocytes is situated in the cytosol. (2) The rest is not mitochondrial, but probably peroxisomal. (3) Of the total catalase activity in control mouse hepatocytes, 5–10% is found in the cytosol. (4) Treatment of mice with clofibrate increases the total hepatocyte activity of soluble epoxide hydrolase 4-fold, but does not influence the relative distribution of this enzyme between cytosol and peroxisomes. (5) The total catalase activity is increased 3.5-fold by clofibrate treatment and 15–35% of this activity is shifted from the peroxisomes to the cytosol.