BACKGROUND:One of the outcomes positively associated with dioxin exposure in humans is type 2 diabetes. OBJECTIVES:This study was conducted in order to find the molecular biological evidence for the diabetogenic action of dioxin in adipose samples from Vietnam veterans. METHODS:We obtained 313 adipose tissue samples both from Vietnam veterans who were exposed to dioxin (Operation Ranch Hand) and from comparison veterans who served in Southeast Asia with no record of dioxin exposure. We conducted quantitative reverse-transcribed polymerase chain reaction studies on selected marker mRNAs from these samples. RESULTS:We found the most sensitive and reliable molecular indicator of dioxin-induced diabetes to be the ratio of mRNA of glucose transporter 4 (GLUT4) and nuclear transcription factor kappa B (NFkappaB), a marker of inflammation. This ratio showed significant correlations to serum dioxin residues and to fasting glucose among those in the Ranch Hand group and, surprisingly, even in the comparison group, who have low levels of dioxin comparable to the general public. Such a correlation in the comparison group was particularly significant among those with known risk factors such as obesity and family history of diabetes. CONCLUSIONS:These results show that the GLUT4:NFkappaB ratio is a reliable marker for the diabetogenic action of dioxin, particularly at very low exposure levels that are not much higher than those found in the general public, implying a need to address current exposure levels.

The effect of single intraperitoneal injection of 115 μg/kg of TCDD (i.e., approximately 1/2 of LD50) to male C57BL/6 mice on the liver mRNA expression changes of several growth factor related genes was assessed at 3 h, 24 h, 10 days, and 30 days posttreatment. The results revealed that the most consistently elevated mRNAs during the entire test period were those of c‐Src, TGFα, and PDGFa. In contrast, those observed to be consistently suppressed were mRNAs for EGF receptor (EGFR), Ki‐Ras, SAPKK, Sp‐1, C/EBPβ, and NFkB. Elevation of mRNAs for TGFβ and STAT3 was observed only on day 10 and day 30. To assess the role of c‐Src in the above action of TCDD, we conducted a parallel study with congenic C57BL/6 male c‐src −/− mice. The results showed that in scr −/− mice the effect of TCDD was less in the case of mRNA expression of PDGF AA , STAT3, C/EPBβ, NMT‐1, and AP‐2γ in addition to c‐src as compared to scr +/+ mice. Those affected least by the absence of c‐Src were SAPKK, and surprisingly, EGF receptor mRNAs, both of which were consistently downregulated in both strains. In most of the other cases, the extent of TCDD‐induced changes were generally less pronounced in src −/− mice as compared to +/+ mice. These observations support the notion that c‐Src is an important mediator of the effects of TCDD on TGFα, PDGF AA , and C/EBPα, β. © 2003 Wiley Periodicals, Inc. J Biochem Mol Toxicol 17:305–315, 2003; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10096

Dioxin is known to cause many toxic effects that vary greatly in different tissues, ages, genders, and species. In this review, an attempt has been made to sort out major signaling pathways involved in the expression of the toxicities of dioxin. The major strategy adopted in analyzing its major signaling pathways is to view the toxic actions of dioxin as the result of the Ah receptor-mediated expression of a major cellular emergency stress response signal. Evidence pointing to the similarities between the symptoms of poisoning by dioxin and those produced by chronic administration of typical stressors, particularly lipopolysaccharides (LPS), bacterial endotoxins, has been assembled and analyzed. The common symptoms are wasting syndrome, atherosclerosis, fatty liver, and thymic atrophy. On the other hand, oxidative stress caused by cytochrome P450 induction is one of the typical stresses of dioxin poisoning, but not LPS poisoning. One of the major means through which dioxin triggers stress responses via "stress-activated kinase pathways" is stimulation of the cellular production of cytokines/autocrines, particularly growth factors. In the case of hepatocytes for instance, transforming growth factor-α plays a pivotal role in the dioxin-induced activation of the epidermal growth factor receptor and the extracellular signal-related kinase pathway, which acts as a signal to suppress apoptosis induced by cellular stress. These observations as well as additional experimental data support the idea that one of the major functions of the Ah receptor could be the elicitation of cellular stress response reactions. Another key point in understanding the toxic action of dioxin is that, unlike other cases of stressors, dioxin signaling becomes chronically sustained because of its extreme persistence in the human body, its half-life of 7–10 years, and its selective accumulation in fatty target tissues.

Based upon our initial observations that 1,1,1-trichloro-2,2-bis(p-chlorophenyl)-ethane (p,p′-DDT) induces a concentration-dependent increase in 3T3-L1 adipocyte differentiation, the mechanism of the p,p′-DDT-induced adipocyte differentiation was studied, using 3T3-L1 and 3T3-F442A cells. Since, it is known that the differentiation of the 3T3-L1 adipocyte cell line involves the induction of the transcription factors CCAAT enhancer binding protein β (C/EBPβ), peroxisome proliferator-activated receptor γ (PPARγ), and C/EBPα, the possible role of these factors in p,p′-DDT-induced adipocyte differentiation had to be examined. It was found that p,p′-DDT-treated 3T3-L1 cells showed a concentration-dependent increase in the nuclear levels of both PPARγ and C/EBPα protein. On the other hand, treatment with p,p′-DDT (20 μM) did not affect the expression pattern of C/EBPβ protein during differentiation. Gel shift analysis of nuclear proteins for binding to the C/EBP recognition site of DNA showed an increase in binding activity at day 2 of differentiation in p,p′-DDT-treated cells. Supershift analysis revealed that this rise was caused mainly by a dramatic increase in the abundance of the C/EBPα–DNA complex. Similar increases were observed at days 4 and 7 after the induction of differentiation. Tumor necrosis factor α induced a strong inhibition of adipocyte differentiation, which was reversed by co-treatment with troglitazone, an activator of PPARγ. p,p′-DDT was unable to reverse the inhibitory effect of tumor necrosis factor α on adipocyte differentiation in 3T3-L1 cells. 3T3-F442A is another preadipocyte cell line that can be induced to differentiate into adipocytes in the presence of insulin and fetal bovine serum. p,p′-DDT (20 μM) induced an alteration in the morphology of these cells at day 2 after the induction of differentiation. These cells however, were unable to become fully differentiated adipocytes. These data showed, therefore, the ability of p,p′-DDT to alter the differentiation process of adipocyte cell lines through the modification of transcription factors regulating this event.

The effects of in vivo administration of the cyclodiene tumor promoter heptachlor epoxide on mouse liver protein kinase C were studied in male B6C3F1 mice by protein kinase C activity assays and Western blotting under conditions known to increase the incidence of hepatocellular carcinoma because protein kinase C is thought to be critical in phorbol ester-induced tumor promotion. Under these test conditions, 20 ppm dietary heptachlor epoxide for 1-20 days increased cytosolic and decreased particulate total protein kinase C activities, while 10 ppm had no effect. Further, total cytosolic and particulate protein kinase C activities were decreased within 1 hour by 10 mg/kg intraperitoneal (i.p.) heptachlor epoxide. Western blotting showed that conventional protein kinase C alpha and beta isoforms were unaffected by heptachlor epoxide. Particulate novel protein kinase C epsilon, however, was selectively down-regulated by 1, 10, and 20 ppm dietary heptachlor epoxide, whereas the cytosolic isoform was decreased by 1 and 10 ppm heptachlor epoxide for 10 days. The high-dose treatment for 24 hours also decreased particulate novel protein kinase C epsilon but increased the cytosolic titer. These results demonstrate that this isoform is unique in its sensitivity to heptachlor epoxide. Activator protein-1 DNA binding, a critical factor in tumor promotion, was substantially increased at 3 and 6 hours with 3.7 mg/kg (i.p.) heptachlor epoxide and at 3 and 10 days with 20 ppm dietary heptachlor epoxide. The effects of heptachlor epoxide on protein kinase C and activator protein-1 are similar to those caused by phorbol ester treatments and correlate well to heptachlor levels found to induce tumors in mice. However, heptachlor epoxide did not initially activate protein kinase C with in vivo treatments or with in vitro treatments of a plasma membrane fraction aimed at demonstrating direct activation, as has been shown for phorbol esters. The ability of heptachlor epoxide to down-regulate particulate novel protein kinase C epsilon correlates to dosages used in in vivo tumor promotion studies. However, this may represent a negative feedback response rather than a causative effect. (C) 2001 John Wiley & Sons, Inc.

Studies were conducted to determine intracellular hepatic signaling pathways associated with estrogen exposure in rainbow trout. These studies were initiated as a first step in elucidating potential signal transduction pathways associated with exposure to environmental estrogens in fish. Studies include analysis of intracellular pathways that are dependent upon estrogen receptor signaling including vitellogenin and choriogenin biosynthesis, and key pathways thought to act through specific kinase cascades. Exposure of 5 mg/kg estradiol to juvenile rainbow trout resulted in a strong and specific ‘estrogenic’ response as measured by the de novo synthesis of vitellogenin and choriogenin in plasma of both male and female trout. Estrogen receptor levels were quantified by both E2 binding and mobility shift assays and demonstrated a significant increase in treated fish. Protein kinase A activity was consistently decreased in estrogen treated males and females, whereas juvenile female trout demonstrated a consistent increase in MAP kinase activity with a concatenate rise in the DNA binding activity of AP-1. Further studies are underway in our laboratories to address whether these cellular responses are estrogen receptor dependent and/or independent signaling events.

To investigate the role of the heterocyclic moieties of nitrogen-containing phenyl heterocyclic compounds (PHCs) in interaction with γ-aminobutyric acid (GABA)-gated chloride channels, diverse classes of PHCs were examined for their ability to inhibit the specific binding of [3H]4′-ethynyl-4-n-propylbicycloorthobenzoate (EBOB), a noncompetitive GABA antagonist, to housefly head and rat brain membranes. PHCs that inhibited [3H]EBOB binding include pyrazoles; 1H-1,2,3-triazoles; a 1H-1,2,4-triazole; 1,3,4-oxadiazol-2(3H)-ones; a 1,3,4-oxadiazole-2(3H)-thione; 1,2,4-oxadiazoles; a 1,2,4-thiadiazole; thiazoles; a 4(3H)-pyrimidinone; and a 2,4(1H,3H)-pyrimidinedione. An analogue (1) of the pyrazole insecticide fipronil, bearing an SCF3 group in place of the S(O)CF3, was found to be the most potent inhibitor with IC50s of 7.55 and 177 nM in housefly head and rat brain membranes, respectively. 3-(2,6-Dichloro-4-trifluoromethyl phenyl)-5-t-butyl-1,3,4-oxadiazol-2(3H)-one exhibited the highest selectivity for housefly GABA receptors versus rat receptors (IC50 rat/IC50 fly >204). PHCs that exhibited a comparable selectivity include a pyrazole and a 1H-1,2,3-triazole. Interaction of 16 selected PHCs with rat brain GABA-gated channels was also examined using [3S]t-butylbicyclophosphorothionate (TBPS), a radioligand for the mammalian noncompetitive antagonist site. A plot of pIC50s of 12 PHCs revealed a close correlation (r = 0.93) between their potency in inhibiting [3H]EBOB and [35S]TBPS binding. Scatchard analyses of the inhibition of [35S]TBPS binding by 1 suggested a competitive-type inhibition. A plot of the potency of 14 PHCs in inhibiting [3H]EBOB binding to housefly head membranes against their piperonyl butoxide-synergized insecticidal effect on German cockroaches yielded a close correlation (r = 0.89), with the exception of five triazoles and a 2,4(1H,3H)-pyrimidinedione. Although several selected PHCs also inhibited the specific binding of [3H]batrachotoxinin A 20-α-benzoate, a tritiated analogue of the sodium channel activator batrachotoxinin, to synaptosomes and membranes prepared from mouse brains, housefly heads, and American cockroach nerve cords, the concentrations required were higher than those of standard compounds producing significant effects on this system. The results demonstrate that a variety of five- and six- membered, nitrogen-containing heterocyclic compounds, bearing a 2,6-dichloro-4-trifluoromethyphenyl or 2,4,6- trichlorophenyl group, interact with ionotropic GABA receptors. Several PHCs display higher affinities for housefly GABA receptors and high selectivity as compared to rat GABA receptors. PHCs' insecticidal activity is mediated by their interaction with GABA-gated chloride channels.

To study the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on nuclear protein phosphorylation activities, male guinea pigs were treated in vivo with a single 1 μg/kg i.p. injection of TCDD, and the state of protein kinases and phosphatases in the nuclei of the hepatocytes was examined after 1, 10, and 40 days. TCDD was found to cause a rise in nuclear protein tyrosine kinase on day 1, and to a lesser extent on day 10, but this effect diminished almost completely on day 40. TCDD also caused a reduction in nuclear casein kinase II (CKII) activity at all time points. To study the biochemical events taking place at the early stage of the action of TCDD, a short-term in vitro model system was established using explant liver tissues maintained in tissue culture medium. It was found that TCDD caused a rapid reduction of the activity of nuclear CKII with an accompanying increase in the cytosol. Such changes in protein phosphorylation activities were also accompanied by an increase in the DNA binding activity of activator protein 1 (AP-1). The effect of TCDD on nuclear proteins binding to the c-Myc response element DNA was, on the other hand, biphasic: an initial increase of protein binding to the c-Myc response element was followed by suppression. To test the hypothesis that some of the above changes were caused by TCDD-induced changes in protein kinase activity, nuclear proteins isolated from hepatocytes of in vivo treated guinea pigs were incubated with exogenously added Mg2+ and ATP under cell-free conditions. The results showed that this in vitro phosphorylation treatment exacerbated this tendency of increased AP-1 and decreased c-Myc binding to their respective response element DNAs, indicating that kinases and phosphatases present in the isolated nuclear protein preparation were active and capable of modifying protein binding to DNA. Such effects of Mg2+ and ATP on AP-1 were blocked by heparin, indicating that CKII plays an important role in transducing the signal of TCDD into the nucleus.

Several studies have shown that some organochlorine compounds act like estrogen in certain animals and in vitro cell culture systems, and therefore, there is a possibility that they could promote the process of tumorigenesis in breast cancer cells. In our previous study, two representative organochlorines, 1,1,1-trichloro 2-o-chlorophenyl-2′-p-chlorophenyl ethane (o,p′-DDT) and β-1,2,3,4,5,6-hexachlorocyclohexane (β-HCH), were found to directly activate the protein tyrosine kinase of Neu (c-erbB-2 proto-oncogene product) immunoprecipitates isolated from MCF-7 breast cancer cells. In the current study, we also found that 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) at 1 nM and α-HCH isomers at 100 nM could also significantly activate protein tyrosine kinase of Neu immunoprecipitates in a cell-free system. We also found that organochlorines result in an increase of Neu protein tyrosine kinase after intact cell treatment in estrogen-depleted medium. This Neu kinase activation by β-HCH (100 nM) was blocked when the cells were pretreated with Neu mRNA antisense oligonucleotide (p < 0.07, Student's t-test). Endogenously added α-, β-, and γ-HCH, o,p′-DDT, 2,2′-dichlorobiphenyl (2,2′-PCB), and 2,4,5-T at 100 nM were found to promote foci formation in postconfluent cultures of this cell line. This stimulatory effect caused by 17β-estradiol, o,p′-DDT, and β-HCH on foci formation was inhibited by coincubation with Neu monoclonal antibody (p < 0.05). Those two events induced by organochlorines (i.e., Neu kinase activation and foci formation) seemed causally correlated. © 1999 John Wiley & Sons, Inc. J Biochem Toxicol 13: 296–302, 1999

To evaluate the extent of organochlorine pollution in the Sierra Nevada ecosystem, residues of certain organochlorines in lake trout and Kokanee fish from Lake Tahoe, an alpine lake located between the Sierra Nevada Mountain Range and the Carson Range of California and Nevada, were analyzed. Multiresidue analysis in fish muscle revealed wet weight concentrations of total polychlorinated biphenyls (PCBs) in the range 18 to 430 ppb and ofp,p′-DDE in the range 5 to 85 ppb in the two fish species studied. In one large lake trout sample (6.6 kg), which was studied in more detail as compared with others, residue levels of PCB (267 ppb), toxaphene (154 ppb), a chlordane mixture (78 ppb), and a DDT mixture (154 ppb) were found in muscle. Full spectra of specific PCB congeners andp,p′-DDE were obtained from fish fat tissues and their identities were confirmed using gas chromatography/mass spectrometry (GC-MS). The results of total PCB analysis indicated that residues found in fish consisted mostly of moderately (tri- to tetrachloro-) to highly (penta- to heptachloro-) chlorinated biphenyls. For all fish residues analyzed, the best match to PCB residue profiles was with Aroclor 1260 or 1262.

Plants are known to activate specific defense mechanisms in response to invasion by pathogens and environmental stresses. We examined the possibility of utilizing the changes in the amounts of protein kinase C (PKC) and its associated components of this signal transduction pathway as a biomarker of exposure of plants to the stress factors, using young rice plants as a model and a Western blotting method as the experimental tool. Preliminary studies have shown that PKC is a consistently more sensitive marker of exposure to a variety of environmental stress factors as compared to phospholipase C (PLC) or G-protein. The titer of PKC increased as a result of exposure to herbicides, low concentrations of copper, fungicides and other chemicals. The same trend was observed when rice plants were stressed by severe physical treatment such as broken stems, deprivation of sunlight and transplantation. On the other hand, PKC levels decreased upon exposure to high winds and high concentration of copper. The most drastic rise in PKC was observed when rice plants were inoculated with the rice blast fungus Pyricularia oryzae. As expected, several plant protectants against the fungal invasion also induce a rise in PKC.

In this report, we studied the effect of phosmidosine, a proline-containing nucleotide on the serum-induced cell cycle progression in human lung fibroblast WI-38 cells. Phosmidosine suppressed S-phase entry and arrested cell cycle progression at the G1 phase. In serum-stimulated cells, phosmidosine did not affect the activation of the mitogen-activated protein kinase cascade. However, phosmidosine inhibited hyperphosphorylation of retinoblastoma (RB) protein by RB-kinases such as cyclin-dependent kinase 4 and cyclin-dependent kinase 2, probably as a result of the inhibition of cyclin D1 expression. Furthermore, in tsFT210 cells, a temperature-sensitive cdc2 mutant isolated from the mouse mammary carcinoma cell line FM3A, phosmidosine, irreversibly inhibited the cell cycle progression at G1 without affecting the G2 to M transition. Phosmidosine acts at an earlier point in G1 compared with mimosine or aphidicolin, well-known cell cycle blockers at the G1-S boundary. Taken together, phosmidosine arrested cells at a specific point between the start point and restriction point in G1 and is a useful drug that may contribute to the understanding of the regulatory mechanisms of G1 progression.

Complementary DNA sequences of genes encoding calmodulin, partial structures of calmodulin-dependent protein kinase II (CaM-kinase II) and an L-type-like calcium channel al subunit (IVS5-IVS6-EF hand region) were identified and compared between susceptible and kdr strains of German cockroach, Blattella germanica. For this purpose, polymerase chain reactions (PCR) were used to obtain their sequences using cDNA from poly(A) + RNA isolated from their heads and thoraces. No mutation differences were found in all three sequences of calcium-regulating proteins between susceptible and strain. Northern blot analysis, however, showed reduced expressions of CaM-kinase II mRNA in two kdr strains. Western blot analysis with an antibody preparation against CaM-kinase II on protein levels confirmed the above strain difference in the titer of this enzyme. In contrast, the levels of calmodulin as well as that of an L-type-like calcium channel gene expression were not different between susceptible and kdr strains.

Dioxin is said to be the most toxic man made chemical ever known. Yet, the toxic action mechanisms for this group of chemicals, other than initial binding to the Ah receptor and subsequent transcriptional activation of certain genes, remain largely unknown. In this article we describe the results of our research efforts over the past 15 years to show why TCDD causes a spectacular rise in the activity of protein tyrosine kinases and how such a phenomenon is related to its expression of toxicity. The pursuit of the former question led to the discovery of c-Src kinase (pp60(c-src)), being the kinase directly associated with the Ah receptor and being the one immediately activated upon TCDD binding to this receptor. More recently we could test this hypothesis in vivo by studying the effects of a single dose of TCDD (115 mu g/kg i.p.) on c-src knockout mice in comparison with the wild type littermates. We found that many of the hallmark effects of TCDD, such as body weight loss, reduction in the weight of adipose tissue, pancreas and heart, thymic involution, and fatty infiltration of the liver, etc., were absent from these c-src deficient mice, while the enlargement of liver (hepatomegaly), induction of cytochrome P450, formation of liver edema, etc., were not affected at all by this genetic manipulation, c-Src kinase appears to play a crucial role in the expression of the toxic effects of TCDD.

A gene fragment belonging to the cytochrome P-450 superfamily has been cloned and identified from stationary cultures of the filamentous fungus Phanerochaete chrysosporium by reverse transcriptase (RT)-PCR, A set of degenerate primers homologous to highly conserved regions of known cytochrome P-450 sequences were used for initial RT-PCRs, Individual PCR products were cloned, sequenced, and identified as those belonging to the cytochrome P-450 superfamily based on amino acid sequence homologies and the presence of the highly conserved heme binding region. The nucleotide sequence of a single cDNA clone indicated the presence of an open reading frame encoding a partial cytochrome P-450 protein of 208 amino acids, Comparisons of the deduced amino acid sequence of the partial protein to other known cytochrome P-450 sequences indicate that it is the first member of a new family of cytochrome P-450s, designated CYP63-1A. Northern blot analysis suggests that CYP63-1A is expressed under both nitrogen-rich and nitrogen-deficient culture conditions and thus not under the same regulatory constraints as the well-studied lignin and manganese peroxidases, Western blot analyses using antibodies raised to the heme binding region of CYP63-1A indicate that the protein has a molecular mass of approximately 44,000 Da.

We have shown previously that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) under cell-free conditions causes a significant rise in protein tyrosine kinase activity of cytosol from male guinea pig adipose tissue, and that such an effect of TCDD is Ah-receptor dependent. In the present study, we obtained evidence indicating that c-Src protein kinase is the protein kinase activated by TCDD and that this kinase is associated specifically with the Ah-receptor-complex proteins in cytosol from adipose tissue and liver of guinea pig and liver of C57B1/6] mouse, and in NIH 3T3 mouse fibroblast cells. Here, we present evidence that c-Src protein is functionally attached to the Ah-receptor (AhR) and is specifically activated upon ligand binding. This conclusion is based on several lines of evidence: (a) TCDD caused activation of protein tyrosine kinase activity when administered directly to purified Ah-receptor immunoprecipitate; (b) this stimulatory effect of TCDD was abolished when the cytosol was immunodepleted of c-Src protein or Ah-receptor protein by preincubating with anti-c-Src or anti-Ah-receptor antibody, followed by the addition of TCDD to the remaining portions of cytosol; (c) when Ah-receptor immunoprecipitate was incubated with TCDD, and the kinase(s) released to the super-natant was analyzed on autoradiography of two-dimensional (2D) electrophoresis, 32P-labeled c-Src protein was recognized; (d) the same 32P-labeled-phosphoprotein with Mr = 60 kDa and pI = 6.1 was found in the immunoprecipitate with anti-c-Src antibody on 2D autoradiograms; (e) this same phosphoprotein disappeared when the supernatant of the Ah-receptor immunoprecipitate was immunodepleted of c-Src protein by anti-c-Src antibody; and (f) a structure-activity relationship study with TCDD and three dioxin-congeners revealed a rank order for their potency in activation of c-Src kinase activity to be identical to that of previously determined toxicity indices: i.e. TCDD>1,2,3,7,8-pentachlorodibenzo-p-dioxin (1,2,3,7,8-PCDD) > 1,2,4,7, 8-pentachlorodibenzo-p-dioxin (1,2,4,7,8-PCDD)>2,7-dichlorodibenzo-p-dioxin (2,7-DCDD). Consistent with these results, TCDD-induced c-Src kinase activity was abolished when c-Src immunoprecipitate's suspension was preincubated with 0.1 or 1 μM α-naphthoflavone (AhR blocker) for 10 min prior to the addition of TCDD. In addition, pretreatment of 3T3 fibroblast cells with 3-methylcholanthrene abolished TCDD-induced c-Src kinase activity in AhR-immunoprecipitate. We conclude that c-Src protein kinase is associated specifically with the AhR complex along with hsp90 in the cytosol of these cells and that upon ligand binding to the Ah-receptor subunit, c-Src is activated and released from the complex.

Recent studies have shown that cultures of white rot fungi not favoring the production of lignin and manganese peroxidases are effective in degrading certain xenobiotics. In this study we have used endosulfan as a model xenobiotic to assess the enzymatic mechanisms of pesticide metabolism under ligninolytic (nutrient-deficient) and nonligninolytic (nutrient-rich) culture conditions. Rapid metabolism of this chlorinated pesticide occurred under each nutrient condition tested, However, the extent of degradation and the nature of the metabolic products differed for nutrient-deficient and nutrient-rich media, The pathways for endosulfan metabolism were characterized by analysis of the fungal metabolites produced, The major endosulfan metabolites were identified by gas chromatography-electron capture detection and gas chromatography-mass spectrometry as endosulfan sulfate, endosulfan diol, endosulfan hydroxyether, and a unknown metabolite tentatively identified as endosulfan dialdehyde. The nature of the metabolites formed indicates that this organism utilizes both oxidative and hydrolytic pathways for metabolism of this pesticide. Piperonyl butoxide, a known cytochrome P-450 inhibitor, significantly inhibited the oxidation of endosulfan to endosulfan sulfate and enhanced hydrolysis of endosulfan to endosulfan diol. We suggest that the metabolism of endosulfan is mediated by two divergent pathways, one hydrolytic and the other oxidative. Judging by the inactivity of extracellular fluid and partially purified lignin peroxidase in metabolizing endosulfan, we conclude that metabolism of this compound does not involve the action of extracellular peroxidases.

This study examined the differences in mechanisms of toxicity when adipose cells from males and females were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Glucose uptake by adipose tissue in vitro was decreased significantly in male guinea pigs within 1 d of intraperitoneal injection of TCDD, but there was no significant effect in females, even at 28 d after treatment. A similar difference between male and female guinea pigs was detected in the effect of TCDD on lipoprotein lipase (LPL) activity, except that a significant decrease in LPL activity was observed 28 d after treatment. Experiments with adipose tissue expiants from untreated guinea pigs and macaques revealed similar gender differences in the effect of TCDD in vitro on glucose uptake and LPL activity. Both time-course studies and dose-response studies with TCDD in vitro confirmed the greater sensitivity of male tissues to TCDD toxicity. TCDD induced lipid peroxidation in the adipose tissues of male guinea pigs, while it had no effect in females. 3H-TCDD binding affinity studies in adipose expiant tissues showed that tissues from male guinea pigs and monkeys had a higher binding capacity for TCDD than female tissues. TCDD induced a significant reduction in nuclear protein phosphorylation and an increase in cytosolic protein phosphorylation in adipose tissue from male guinea pigs; the effects in female tissues were opposite: nuclear protein phosphorylation increased and cytosolic protein phosphorylation decreased. In a cell-free system in the absence of the nucleus, adipose tissues from male guinea pigs and monkeys responded to TCDD with a rapid stimulation of tyrosine kinase activity but female tissues from both species had a significantly lower and slower response. TCDD induced the DNA binding of AP-1 in adipose tissues of male guinea pigs, but in female tissues TCDD reduced the DNA binding of AP-1. In summary, the results of this study demonstrate gender differences in the response of nonreproductive cells to TCDD. Some of these differences involve different mechanisms of toxicity in both the cytoplasmic and nuclear compartments of the cell.