Progressive resistance to apoptosis in a cell lineage model of human proliferative breast disease.

BACKGROUND: Proliferative breast disease (PBD) may increase a woman's risk of developing breast cancer, perhaps by decreasing cellular sensitivity to apoptosis. To determine whether resistance to apoptosis develops during PBD, we investigated apoptosis initiated through the Fas pathway in a series of cell lines that recapitulates the morphologic changes of PBD in nude/beige mice. METHODS: The series of cell lines used was MCF-10A cells (parental preneoplastic human breast epithelial cells), MCF-10AT cells (transformed with T(24) Ha-ras), and MCF-10ATG3B cells (derivative cells that progress to carcinoma). Fas-mediated apoptosis, induced when a Fas monoclonal antibody bound to and activated the Fas receptor on these cells, was assessed morphologically and by flow cytometry. Levels of proteins involved in Fas-mediated apoptosis and cleavage of poly(adenosine diphosphate-ribose) polymerase (PARP), an end product of caspase activation, were determined by immunoblotting. Bcl-2 and Bax heterodimerization was examined by coimmunoprecipitation. All statistical tests were two-sided. RESULTS: Sensitivity to Fas-mediated apoptosis decreased with the tumorigenic potential of cells: MCF-10A cells were extremely susceptible, MCF-10AT cells were less susceptible, and MCF-10ATG3B cells were resistant. The percentage of apoptotic cells declined, from 24% to 8% to 6%, respectively. All lines produced Fas ligand (FasL) and had comparable levels of Fas receptor, FasL, Fas-associated death-domain protein, and caspases 3 and 6. Levels of caspase 8 were similar in MCF-10A and MCF-10AT cells but about 30% lower in MCF-10ATG3B cells (P>.01 but <.05). Levels of caspase 10 were about 20% lower in MCF-10AT cells (P>.005 but <.01) and about 59% lower in MCF-10ATG3B cells than in MCF-10A cells (P>.01 but <.05). PARP cleavage was detected in MCF-10A and MCF-10AT cells but not in MCF-10ATG3B cells. Levels of Bax, Bid, and Bak proteins were similar in all lines, but levels of Bcl-2 were lower in MCF-10AT and MCF-10ATG3B cells than in MCF-A cells, and Bcl-2-Bax heterodimerization progressively declined in the series. CONCLUSION: Resistance to Fas-mediated apoptosis appears to develop progressively in the MCF-10AT cell series.

Role of P450IIE1 in the metabolism of 3-hydroxypyridine, a constituent of tobacco smoke: redox cycling and DNA strand scission by the metabolite 2,5-dihydroxypyridine.

The metabolism of 3-hydroxypyridine, a significant constituent of tobacco smoke, to 2,5-dihydroxypyridine has been characterized in hepatic microsomes and in the reconstituted enzyme system using purified forms of P450. The redox cycling activity of the metabolite and its ability to damage DNA in vitro have been examined. Pyridine-induced microsomes, which contain elevated levels of P450IIE1 (Kim et al., J. Pharmacol. Exp. Ther., 246: 1175-1182, 1988), catalyzed an 8-fold increase in the production of 2,5-dihydroxypyridine, relative to control, which showed biphasic kinetics. Pyridine-induced rabbit hepatic microsomes exhibited a Vmax of 5.9 nmol 2,5-dihydroxypyridine/min/mg protein and a Km value of 110 microM. In contrast, phenobarbital- and isosafrole-induced microsomes had Vmax values of 2.5 and 1.2 nmol/min/mg protein and Km values of 590 and 134 microM, respectively. Pyridine-induced rat hepatic microsomes also exhibited elevated catalytic activity toward the hydroxylation of 3-hydroxypyridine, with an 8-fold increase in Vmax (2.74 nmol/min/mg protein) relative to uninduced rat hepatic microsomes (Vmax = 0.34 nmol/min/mg protein). In the reconstituted system, cytochrome P450IIE1 displayed the greatest activity in the production of 2,5-dihydroxypyridine of the major forms of rabbit P450 examined. P450IIE1 was 34-fold more active than P450IIB1 and 12-fold more active than P450IA2 in the production of 2,5-dihydroxypyridine. The redox cycling activity of 2,5-dihydroxypyridine has been characterized. The rate of NADPH oxidation in the presence of 0.5 mM 2,5-dihydroxypyridine was stimulated approximately 4-fold (69.2 nmol NADPH oxidized/min/mg protein), relative to control (16 nmol/min/mg protein). 2,5-Dihydroxypyridine at 0.5 and 1.0 mM produced a 12- and 17-fold increase, respectively, in the rate of superoxide anion production compared to control, as monitored by the SOD-inhibitable reduction of acetylated cytochrome c. 3-Hydroxypyridine alone failed to increase the rate of superoxide production. Inclusion of reduced glutathione in the incubation resulted in a pronounced decrease in the 2,5-dihydroxypyridine-stimulated rate of cofactor oxidation and superoxide production. The ability of 2,5-dihydroxypyridine to damage DNA was assessed by monitoring phi X-174 DNA strand scission. The band intensity of the supercoiled form of DNA, when incubated with 1 mM 2,5-dihydroxypyridine, decreased substantially, with a concomitant increase in intensity of the band associated with the open circular form of DNA. The change in phi X-174 DNA topology produced by 2,5-dihydroxypyridine was accelerated in a dose-dependent manner, with an estimated EC50 of approximately 60 microM.(ABSTRACT TRUNCATED AT 400 WORDS)

Xenobiotic-modulated expression of hepatic glutathione S-transferase genes in primary rat hepatocyte culture.

CYP 2B1/B2 and 1A1 expression in primary rat hepatocytes plated on a substratum of Vitrogen using Chee's Essential Medium has been reported to be responsive to xenobiotic treatment (Jauregui, H.O., Ng, S.F., Gann, K.L. and Waxman, D.J. (1991) Xenobiotica 21, 1091-1106). Class alpha, mu and pi glutathione S-transferase (GST) gene expression in response to xenobiotic treatment using this primary hepatocyte culture system was examined and the results compared with those obtained for P4502B1/B2 and 1A1 expression. Cytosolic GST activity decreased approx. 75% during the first 48 h of culture relative to freshly isolated hepatocytes and subsequently, increased, attaining a level at 96 h that was 134% of the activity at 48 h post-plating. Treatment of the hepatocyte cultures with phenobarbital (2 mM) or 3-methylcholanthene (5 microM) for 24, 48, or 72 h, beginning 24 h after plating, resulted in significant increases in glutathione S-transferase activity relative to control, with maximal increases of 158 and 164% measured at 72 h following phenobarbital or 3-methylcholanthrene treatment, respectively. SDS-PAGE analysis of cytosolic proteins showed a substantial increase in the intensities of protein bands migrating in the region of the GSTs following phenobarbital, beta-naphthoflavone or 3-methylcholanthrene treatment. Immunoblot analysis of cytosolic fractions using affinity-purified class-specific GST IgGs confirmed that alpha, mu and pi-class GST isozymes were elevated approx. 1.5- to 2-fold following phenobarbital, or beta-naphthoflavone treatment; 3-methylcholanthrene was less effective in enhancing GST expression in cultured hepatocytes as compared to phenobarbital or beta-naphthoflavone. Although GST pi was below the limit of detection in freshly-isolated hepatocytes, enhanced expression of this form was observed in untreated hepatocytes cultured for longer than 72 h. Immunoblot analysis of microsomal fractions revealed that cytochrome P-4502B1/2B2 and 1A1 levels were increased significantly in hepatocyte cultures treated with phenobarbital or 3-methylcholanthrene, respectively, relative to the undetectable levels found in untreated controls. Northern blot analysis of poly(A)+ mRNA isolated from cultures that had been treated with phenobarbital or 3-methylcholanthrene showed an approx. 2- and 4-fold increase in the expression of alpha and pi class glutathione S-transferase mRNAs, respectively, as compared to untreated cells. The level of P-4501A1 or 2B1 mRNA was also markedly elevated following 3-methylcholanthrene or phenobarbital treatment, respectively. The results of this study demonor the first time, that expression of alpha, mu and pi-class glutathione S-transferase genes is effectively modulated in primary yet culture system by different classes of xenobiotics.

Determination of the poly(A) tail lengths of a single mRNA species in total hepatic RNA.

Poly(A) tail length is important in the stability and translation of mRNA. We describe procedures for the rapid and reproducible analysis of poly(A) tail length of a single mRNA species contained in a sample of total hepatic RNA. A short 3' fragment of a specific mRNA is prepared by RNase H digestion of the targeted mRNA region annealed to a short DNA oligonucleotide. The length of the poly(A) tail of the 3' fragment is then determined by running the sample on a polyacrylamide gel, by electrophoretic transfer, by probing with a radiolabeled cDNA and by comparing the size of the detected region with a specific RNA ladder or a DNA ladder.

Effects of altered calcium homeostasis on the expression of glutathione S-transferase isozymes in primary cultured rat hepatocytes.

The effects of altered Ca2+ homeostasis on glutathione S-transferase (GST) isozyme expression in cultured primary rat hepatocytes were examined. Isolated hepatocytes were cultured on Vitrogen substratum in serum-free modified Chee's essential medium and treated with Ca2+ ionophore A23187 at 120 hr post-plating. GST activity increased slightly, albeit significantly, in a concentration-dependent manner in A23187-treated hepatocytes relative to untreated controls. Western blot analysis using GST class alpha and mu specific antibodies showed an approximately 1.6- and 1.5-fold increase in the class alpha, Ya and Yc subunits, respectively, whereas no significant increase (approximately 1.2-fold) in class mu GST expression was observed following A23187 treatment. Northern blot analysis revealed an approximately 5-fold increase in GST class alpha and an approximately 7-fold increase in class mu GST mRNA levels in ionophore-treated hepatocytes compared to untreated cells. Results of the Western and Northern blot analyses of the ionophore-treated hepatocytes were compared with those obtained for tert-butyl hydroperoxide-treated cells. Immunoblot analysis showed a significant increase in the expression of GST class alpha, Ya and Yc subunits, approximately 1.8- and 1.7-fold, respectively, for tert-butyl hydroperoxide-treated hepatocytes as compared to controls, with little or no increase in class mu GSTs. Northern blot analysis showed approximately 3- and 2-fold increases, respectively, in class alpha and mu GST mRNA levels, following the tert-butyl hydroperoxide treatment. The results of the present investigation show that alterations in Ca2+ homeostasis produced by either Ca2+ ionophore A23187 or tert-butyl hydroperoxide treatment of hepatocytes enhanced the expression of GST isozymes in primary cultured rat hepatocytes.

Metabolic and morphologic effects of the antimicrobial agent nitrofurantoin on human erythrocytes in vitro.

We have reported previously that the antimicrobial nitrofurantoin stimulates superoxide production and methemoglobin formation from HbO2 as an isolated hemeprotein and in hemolysates [M. Dershwitz and R. F. Novak, J. biol. Chem. 257, 75 (1982); M. Dershwitz and R. F. Novak, J. Pharmac. exp. Ther. 222, 430 (1982)]. The production of hydrogen peroxide and methemoglobin by nitrofurantoin has been determined in normal erythrocytes in vitro. Hydrogen peroxide production increased 5-fold during a 20-hr incubation in the presence of 840 microM nitrofurantoin, while methemoglobin content increased to over 20% of the total hemoglobin concentration of the cells. Consequent metabolic and morphologic alterations also occurred. Concomitant with nitrofurantoin-stimulated hydrogen peroxide production were time- and concentration-dependent decreases in cellular levels of GSH and ATP, as well as alterations in red cell morphology. Significant differences in GSH and ATP levels between control and nitrofurantoin-treated erythrocytes occurred after 12 hr and proceeded maximally from 18 to 21 hr. After a 21-hr incubation, 840 microM nitrofurantoin caused the cellular GSH and ATP levels to fall 65 and 75%, respectively, while controls exhibited only 29 and 43% decreases in ATP and GSH levels, respectively. Studies on the concentration dependence of such decreases demonstrated that the EC50 values for depletion of GSH and ATP were similar in blood obtained from an individual donor. The EC50 values varied from approximately 10 microM to 100 microM among the various donors whose blood was studied. Incubation of normal red cells with nitrofurantoin also resulted in an increased conversion of red cells to echinocytes as observed by scanning electron microscopy. These metabolic effects, coupled with increased oxidative stress via hydrogen peroxide generation, lend support to the mechanism for nitrofurantoin-induced hemolysis in erythrocytes compromised by certain enzyme deficiencies which result in low basal levels of GSH or diminished rates of GSH synthesis.

Inhibition of cytochrome P4502E1 expression by organosulfur compounds allylsulfide, allylmercaptan and allylmethylsulfide in rats.

Cytochrome P4502E1 (CYP2E1) is active in both detoxication and activation of small organic molecules. The effects of organosulfur compounds including allylsulfide (AS), allylmercaptan (AM) and allylmethylsulfide (AMS) on the expression of CYP2E1 were examined in rats. 4-Nitrophenol, aniline hydroxylase and N-nitrosodimethylamine demethylase activities, the rates of which represent the level of CYP2E1, decreased in hepatic microsomes isolated from rats treated with AS in a time-dependent manner by 45% to 90%, as compared to control. Pyrazine-induced hepatic microsomes exhibited approximately 5-fold increases in CYP2E1-catalysed metabolic activities, whereas the hepatic microsomes obtained after treatment of animals with both AS and pyrazine showed rates comparable to or less than those in control microsomes. AM or AMS suppressed constitutive and pyrazine-inducible levels of CYP2E1 similarly to AS. Immunoblot analyses of hepatic microsomes, using an anti-CYP2E1 antibody, showed that AS, AM and AMS significantly suppressed constitutive levels of CYP2E1 apoprotein after 24, 48 and 72 hr. Time-dependent induction of CYP2E1 by pyrazine was also completely blocked by treatment of animals with AS throughout the experimental period, as evidenced by immunoblot analysis. The levels of CYP2E1 apoprotein in the hepatic microsomes isolated from animals treated with both AM and pyrazine, or with both AMS and pyrazine were comparable to those in control hepatic microsomes at days 1-3 post-treatment. Treatment of rats with each of these organosulfur compounds caused no significant changes in the levels of CYP2E1 mRNA, as assessed by slot and northern blot analyses, suggesting that post-transcriptional regulation may be associated with the suppression of CYP2E1 apoprotein levels. The results of metabolic activities, immunoblot analyses and RNA blot analyses demonstrated that these organosulfur compounds are effective in suppressing constitutive and inducible expression of CYP2E1.

The acute vasculitis of Wegener's granulomatosis in renal biopsies.

The kidney biopsy specimens from five patients with Wegener's Granulomatosis were reviewed in an attempt to characterize the early histological lesion when vasculitis was present. The vascular lesions were found mainly in interlobular-sized arteries. The acute vascular lesions were sparse and focal, mainly localized to the intimal area, and with fibrinoid material and platelets constituting the early infiltrate. The electron microscopy and scanning electron microscopy of the involved areas further emphasized the presence of platelets in the early lesion, and accentuated endothelial alterations occurring along with the intimal infiltrate. Morphologic evidence of immune complex presence was not found.

Cytotoxicity of trichloroethylene and S-(1, 2-dichlorovinyl)-L-cysteine in primary cultures of rat renal proximal tubular and distal tubular cells.

Activities of several glutathione-dependent enzymes, expression of cytochrome P450 isoenzymes, and time- and concentration-dependent cytotoxicity of trichloroethylene (TRI) and S-(1, 2-dichlorovinyl)-L-cysteine (DCVC) were evaluated in primary cultures of proximal tubular (PT) and distal tubular (DT) cells from rat kidney. These cells exhibited cytokeratin staining and maintained activities of all glutathione-dependent enzymes measured. Of the cytochrome P450 isoenzymes studied, only CYP4A expression was detected. CYP4A mRNA and protein expression were higher in primary cultures of DT cells than in PT cells and were increased in DT cells by ciprofibrate treatment. Incubation of cells for 6 h with concentrations of TRI as high as 10 mM resulted in minimal cytotoxicity, as determined by release of lactate dehydrogenase (LDH). In contrast, marked cytotoxicity resulted from incubation of PT or DT cells with DCVC. Addition to cultures of TRI (2-10 mM) for 24 or 72 h resulted in modest, but significant time- and concentration-dependent increases in LDH release. Treatment of cells with DCVC (0.1-1 mM) for 24 h caused significant increases in LDH release and alterations in cellular protein and DNA content. Finally, exposure of primary cultures to TRI or DCVC for 72 h followed by 3 h of recovery caused a slight increase in the expression of vimentin, consistent with cellular regeneration. These studies demonstrate the utility of the primary renal cell cultures for the study of CYP4A expression and mechanisms of TRI-induced cellular injury.

Glutathione S-transferases and gamma-glutamyl transpeptidase in the rat nervous systems: a basis for differential susceptibility to neurotoxicants.

Glutathione and its related enzymes play a major role in the detoxification of toxic chemicals. In rat brain the pattern of distribution of reduced glutathione exhibits cellular heterogeneity, suggesting also the possibility of cellular differences in glutathione conjugating capacity. To understand the potential role of GSH in detoxification of neurotoxicants, the distributions of the glutathione conjugating and metabolizing enzymes, glutathione S-transferase (GST; alpha-, mu- and pi-classes) and gamma-glutamyl transpeptidase (gamma-GT) were determined immunohistochemically in brain, lumbar spinal cord and dorsal root ganglia (DRG) of adult Sprague-Dawley rats using polyclonal antibodies. The influence of tissue fixation on apparent distribution was also examined. Glial cells and neurons throughout the nervous system were only weakly positive with alpha-GST in frozen sections. No immunoreactivity for the alpha-class GSTs was observed in any of the paraformaldehyde-fixed neural specimens examined. In microwave-fixed frozen sections, immunoreactivity to mu-GST was found in astrocytes and neurons throughout the brain and spinal cord, and in the neurons and satellite cells of the DRG. Immunoreactivity for pi-GST was seen in oligodendrocytes but not in astrocytes in any region of the CNS examined. Similarly, satellite cells of the DRG were positive for pi-GST. Neuronal perikarya of the entire neopallium, hippocampus, cerebellum, brainstem, spinal cord and DRG were also positively stained for pi-GST. The differential staining of astrocytes and oligodendrocytes with pi- and mu-GST was unaltered in paraformaldehyde fixed tissues, but the neuronal immunostaining was lost. The ependyma, pia and choroid plexus stained positively with all three GST antibodies regardless of fixation. Gamma-Glutamyl transpeptidase-like immunoreactivity was confined to non-neuronal elements of both central and peripheral nervous systems. Ependymal cells throughout the central nervous systems stained intensely with antibodies directed against gamma-GT. Satellite and Schwann cells of the DRG and glial cells of the spinal cord and brain exhibited moderate to intense immunoreactivity for gamma-GT. The heterogeneous cellular distribution of glutathione and its metabolizing enzymes may reflect cellular differences in capacity for metabolic processing of both endogenous compound and xenobiotics.

Developmental changes in the cellular distribution of glutathione and glutathione S-transferases in the murine nervous system.

The distribution of glutathione (GSH) and glutathione S-transferases (GSTs) in the adult rat brain is cell-type specific, but their cellular distribution in the developing central nervous system is unknown. In the present study, GSH distribution in the mouse nervous system was visualized by mercury orange histochemistry and class-specific GSTs were localized by immunohistochemistry at ages E13 to PN30. Both neuronal and glial progenitor cells stain uniformly positive for GSH at E13. Spinal anterior horn neurons become GSH-negative by E17, at which time neurons and glia in other CNS regions are still GSH-positive. By PN5, most neurons have lost GSH staining and are surrounded by GSH-rich neuropil, ependyma, and vasculature. Olfactory mitral and granule cells, cerebellar granule cells, and dorsal root ganglion (DRG) neurons retain consistently high levels of GSH throughout development and into adulthood. Immunoreactivity to alpha-class GST antisera is not observed in the CNS until PN10, when very weak staining becomes apparent in the pia, ependyma, choroid plexus and neurons throughout the brain and spinal cord. Immunoreactivity to mu-GST is observed in neurons and astrocytes (but not oligodendrocytes), pia, ependyma, and choroid plexus throughout the brain by PN10. pi-GST immunoreactivity is observed in all cells of the embryonic nervous system. Postnatally, it is found in neurons and oligodendrocytes (but not astrocytes) in all regions of the brain and spinal cord as well as in pia, ependyma, and choroid plexus. The neurons and satellite cells of the DRG are immunoreactive to alpha-, mu-, and pi-GST antisera at all time points examined. The developmental changes in the cellular distribution of GSH and GSTs suggest that enzymatic conjugation and antioxidant activities may also be cell specific during brain development.

Effects of mitoxantrone and bisantrene on platelet aggregation and prostaglandin/thromboxane biosynthesis in vitro.

The effects of mitoxantrone and bisantrene on agonist-stimulated platelet aggregation, prostaglandin E2 and thromboxane B2 production were examined and results compared with those produced by indomethacin and acetylsalicylic acid. Both mitoxantrone and bisantrene effectively inhibited collagen-, ADP-, and epinephrine-stimulated platelet aggregation. Collagen (0.54 microgram/ml)-stimulated platelet aggregation was inhibited by 50% at 60 microM mitoxantrone and 8 microM bisantrene. The concentration of drug required for inhibition of platelet aggregation varied inversely with the level of collagen stimulus employed. Mitoxantrone and bisantrene inhibited both the first and second phase of epinephrine-stimulated platelet aggregation. Complete inhibition of the second wave of aggregation was obtained at greater than 160 microM mitoxantrone and 16 microM bisantrene. Mitoxantrone is comparable in potency to acetylsalicylic acid which inhibited the second wave of epinephrine-stimulated aggregation 50% at 160 microM, whereas bisantrene may be compared to indomethacin which produced complete inhibition of aggregation at 16 microM. Production of PGE2 and TXB2 in epinephrine-stimulated platelets was inhibited by both drugs with 50% inhibition of PGE2 production occurring at 12 microM mitoxantrone and 3 microM bisantrene. Thromboxane B2 production was inhibited by 50% at 10 microM mitoxantrone and 5 microM bisantrene. Indomethacin inhibited PGE2 and TXB2 production 50% at 4 microM. Thus mitoxantrone and bisantrene inhibit platelet aggregation and prostaglandin production which may be of significance in metastasis and in prostaglandin-mediated physiologic and immune responses.

Insulin effects on CYP2E1, 2B, 3A, and 4A expression in primary cultured rat hepatocytes.

Although hyperketonemia and/or altered growth hormone secretion caused by diabetes have been implicated in enhanced CYP2E1, 2B, 3A and 4A expression, the effect of insulin on hepatic P450 expression, in the absence of associated metabolic/hormonal alterations, remains unknown. Primary cultured rat hepatocytes have been shown (Zangar et al., Drug Metab. Dispos., 23:681, 1995) to express stable and inducible CYP2E1 mRNA and protein levels, and provide an excellent system for mechanistic examination of the effect of insulin on CYP2E1, 2B, 3A and 4A expression. Maintaining primary rat hepatocytes in culture in the absence of insulin for 48, 72, or 96 h increased CYP2E1 mRNA levels 5-, 11-, and 4-fold, respectively, relative to cells maintained in the presence of the standard concentration of 1 microM insulin. In contrast, CYP2B mRNA levels increased only approximately 2-fold in the absence of insulin, when compared with the presence of 1 microM insulin. CYP2E1 and 2B protein levels were increased 6.7- and 3.8-fold, respectively, in cells cultured for 96 h in the absence of insulin as compared with those cultured in medium containing 1 microM insulin. Concentration-response studies revealed that decreasing the concentration of insulin below 10 nM (i.e. 1 nM, 0.1 nM, no insulin) increased CYP2E1 mRNA levels 4-, 7-, and 11-fold, respectively. In contrast, no such concentration-dependence was observed for CYP2B mRNA expression. As CYP3A and 4A expression is also elevated in diabetic rats, the effects of insulin on these P450s was also examined. CYP3A mRNA levels were unaltered and CYP4A mRNA levels were decreased marginally (approximately 50%) by the absence of insulin relative to levels in cells cultured in the presence of 1 microM insulin over 96 h in culture. The results of this study provide evidence that insulin itself, in the absence of other diabetes-induced metabolic or hormonal alterations, affects CYP2E1 and 2B, but not CYP3A or 4A, expression in primary cultured rat hepatocytes. Furthermore, CYP2E1 expression is differentially regulated by insulin relative to CYP2B, 3A or 4A. This study also demonstrates that decreasing the concentration of insulin in the culture medium provides a method by which CYP2E1 levels can be increased in primary cultured hepatocytes to facilitate mechanistic studies on the regulation of CYP2E1 expression.

Pyridine induction of Sprague-Dawley rat renal cytochrome P4502E1: immunohistochemical localization and quantitation.

Previous research has shown that i.p. injection of rats with pyridine results in a significant increase in immunoreactive renal cytochrome P4502E1 (alcohol-inducible form) in a dose- and time-dependent manner. However, the cellular location of renal P4502E1 in rats was not reported. Thus, it was not known whether the pyridine-induced increase in renal P4502E1 resulted from increased production of the enzyme in cells which normally express P4502E1 or from de novo expression in cells normally devoid of the protein. To address these questions, rats were injected i.p. with either 200 mg pyridine/kg body wt./day for 1, 2, 3, or 4 days (n = 2/group) or injected once with an equal volume of sterile, pyrogen-free saline (control group; n = 2). Kidney tissue samples from saline- and pyridine-exposed rats were processed by light microscopy and were immunochemically stained to detect rat cytochrome P4502E1. Most of the immunoreactive P4502E1 was located within renal cortical epithelial cells lining proximal and distal tubules of the cortex with lesser--but consistent--amounts present in tubular epithelial cells within the inner and outer medulla. Pyridine exposure resulted in a 2-3-fold increase in P4502E1 immunoreactivity in proximal cortical tubules surrounding glomeruli and cortical blood vessels. The results of this study demonstrate a cell-specific distribution of cytochrome P4502E1 within the rat kidney and indicate that pyridine exposure results in a selective induction of immunoreactive P4502E1 in tubule epithelial cells which constitutively express this enzyme. The results of this study provide a morphologic basis for interpreting cell-specific nephrotoxicity due to xenobiotics that are biotransformed to toxic metabolites by renal P4502E1.

A brief review of the anatomy, histology, and ultrastructure of the full-term placenta.

A brief review of the morphologic features of the term placenta is presented using schematic drawings and light and transmission electron microscopic illustrations of individual features. Some areas of current and past controversy are identified regarding placental structure. The review is intended to be an introduction for those examining placentas in surgical pathology laboratories.

Kepone (chlordecone) disrupts adherens junctions in human breast epithelial cells cultured on matrigel.

Perturbations in cell-extracellular matrix (ECM) interactions are a consistent feature of mammary tumors and cells in culture. We have utilized MCF-10ATG3B human breast epithelial cells to examine whether the organochlorine Kepone induces alterations in cell adhesion molecules important to cell-cell and cell-ECM interactions. Kepone effects on the levels and association of proteins involved in adherens junctions or desmosomes were examined using immunoblot analysis and immunoprecipitation. MCF-10ATG3B cells cultured on an ECM of Matrigel form lattice-like structures that are disrupted with 0.1 and 1 microM Kepone. E-cadherin protein levels decreased significantly by approximately 23% and approximately 69% following treatment with 0.1 and 1.0 microM Kepone, respectively, relative to solvent-treated cells. Desmoglein and alpha- and gamma-catenin levels did not vary significantly with Kepone. Beta-catenin protein levels decreased significantly by approximately 37%, 36% and 53% at 0.01, 0.1 and 1.0 microM Kepone, respectively. E-cadherin-gamma-catenin association was disrupted with 0.1 and 1.0 microM Kepone. Thus, Kepone disrupts cellular architecture, specifically E-cadherin-gamma-catenin containing adherens junctions, which may ultimately affect cellular phenotype.

The alcohol-inducible form of cytochrome P450 (CYP 2E1): role in toxicology and regulation of expression.

Cytochrome P450 (CYP) 2E1 catalyzes the metabolism of a wide variety of therapeutic agents, procarcinogens, and low molecular weight solvents. CYP2E1-catalyzed metabolism may cause toxicity or DNA damage through the production of toxic metabolites, oxygen radicals, and lipid peroxidation. CYP2E1 also plays a role in the metabolism of endogenous compounds including fatty acids and ketone bodies. The regulation of CYP2E1 expression is complex, and involves transcriptional, post-transcriptional, translational, and post-translational mechanisms. CYP2E1 is transcriptionally activated in the first few hours after birth. Xenobiotic inducers elevate CYP2E1 protein levels through both increased translational efficiency and stabilization of the protein from degradation, which appears to occur primarily through ubiquitination and proteasomal degradation. CYP2E1 mRNA and protein levels are altered in response to pathophysiologic conditions by hormones including insulin, glucagon, growth hormone, and leptin, and growth factors including epidermal growth factor and hepatocyte growth factor, providing evidence that CYP2E1 expression is under tight homeostatic control.