Immunohistochemical localization of carboxylesterase in the nasal mucosa of rats.

The enzymatic esterase activity of carboxylesterases is integral to the nasal toxicity of many esters used as industrial solvents or in polymer manufacture, including propylene glycol monomethyl ether acetate, dimethyl glutarate, dimethyl succinate, dimethyl adipate, and ethyl acrylate. Inhalation of these chemicals specifically damages the olfactory mucosa of rodents. We report the localization and differential distribution of a 59 KD carboxylesterase in nasal tissues of the rat by immunohistochemistry. Rabbit antiserum against the 59 KD rat liver microsomal carboxylesterase bound most prominently to the olfactory mucosa when applied to decalcified, paraffin-embedded sections of rat nasal turbinates. Within the olfactory mucosa, anti-carboxylesterase did not bind to sensory neurons, the target cell for ester-initiated toxicity; these cells apparently lack carboxylesterase. Instead, the antibody was preferentially bound by cells of Bowman's glands and sustentacular epithelial cells which are immediately adjacent to the olfactory nerve cells. In contrast, non-olfactory tissues (respiratory mucosa and squamous epithelium), which are more resistant to the toxicity of esters, had less carboxylesterase content. The distribution of immunoreactivity correlated well with the distribution of carboxylesterase catalytic activity described elsewhere. These findings help to link the metabolic fate of inhaled esters to the site-specific pathological findings that follow exposure to such chemicals.

Interaction of fluoroethane chlorofluorocarbon (CFC) substitutes with microsomal cytochrome P450. Stimulation of P450 activity and chlorodifluoroethene metabolism.

The abilities of halothane and the fluoroethane chlorofluorocarbon (CFC) substitutes, FC-123, FC-133a, FC-124, FC-134a and FC-125, to stimulate cytochrome P450 activities and 2-chloro-1,1-difluoroethene (CDE) defluorination in hepatic microsomes from phenobarbital-treated rabbits were compared. At 1% (v/v) each, halothane and FC-123 similarly increased the consumption of NADPH and O2 by 300 and 100%, respectively, over that in microsomes without substrate. FC-133a and FC-124 were less effective, increasing NADPH and O2 consumption by 150-200 and 70%. FC-134a and FC-125 were the least effective, increasing NADPH and O2 consumption by only 70 and 50%, respectively. No metabolism of any fluoroethane could be detected under the incubation conditions used. Halothane and FC-123 were most effective in stimulating CDE metabolism with increases of CDE defluorination ranging from 1.5- to 2-fold. FC-133a and FC-124 enhanced CDE oxidation 89 and 74%, respectively, and FC-134a and FC-125 had no effect. While CDE metabolism was enhanced in the presence of the fluoroethanes, no additional NADPH or O2 was consumed when halothane or FC-124 was incubated with CDE compared with incubations containing only halothane or FC-124. Log-log plots of NADPH consumption and CDE metabolism with the olive oil/gas partition coefficients of each fluoroethane showed linear relationships. These data demonstrate that the activity of the fluoroethanes in stimulating P450 activity and CDE metabolism is a function of their lipid solubility, and fluoroethane-enhanced CDE metabolism is related to the ability of these compounds to increase uncoupled P450 activity.

Potentiation of dimethylnitrosamine genotoxicity in rat hepatocytes isolated following ethanol treatment in vivo.

Unscheduled DNA synthesis (UDS), following exposure to dimethylnitrosamine (DMN), was potentiated in cultured hepatocytes isolated following treatment of rats for 14 or 28 days with 20% ethanol/5% sucrose solution. Ethanol treatment was associated with increased UDS, a concomitant increase in hepatic microsomal protein concentration and DMN N-demethylase activity. Increased aniline hydroxylase activity of hepatic microsomes from ethanol-treated rats preceded the measured increase in microsomal protein content or DMN metabolism. The increase in metabolism of DMN in vitro and potentiation of DMN-induced UDS associated with ethanol treatment may contribute to a synergistic effect of ethanol on DMN hepatotoxicity and carcinogenicity. In contrast, ethanol pretreatment did not increase the cytotoxicity of DMN as characterized by enzyme release.

Defluorination of the CFC-substitute 1,1,1,2-tetrafluoroethane: comparison in human, rat and rabbit hepatic microsomes.

1,1,1,2-Tetrafluoroethane (HFC-134a), which lacks ozone-depleting potential, has been selected as a replacement refrigerant for dichlorodifluoromethane (CFC-12) in air-conditioning and chiller applications, and as a propellant for pharmaceutical aerosols. A variety of paradigms using rats and rabbits have shown that HFC-134a has very little toxic potential. To strengthen the prediction of human hazard associated with HFC-134a exposure, we evaluated the rate of metabolism of this halocarbon by human hepatic microsomes relative to similar tissue preparations derived from rats and rabbits. Human microsomes defluorinated HFC-134a in a cytochrome-P-450-catalyzed reaction, common also to rat and rabbit. In absolute terms, the maximal rate of HFC-134a metabolism by human microsomes was very low, showed little interindividual variation among the samples evaluated (1.3 +/- 0.3 nmol F-/mg protein/15 min, mean +/- SD, n = 10), and did not exceed that in rat or rabbit liver microsomes. These findings support the argument that for characterization of HFC-134a toxicity, especially that which may be mediated by products of halocarbon metabolism, laboratory animals are an adequate surrogate for humans.

Leupeptin-mediated alteration of renal phagolysosomes: similarity to hyaline droplet nephropathy of male rats exposed to unleaded gasoline.

alpha 2u-Globulin, a protein of hepatic origin found in the urine of male rats, is accumulated in the kidney cortex during exposure to unleaded gasoline and has been implicated in the development of fuel hydrocarbon-induced nephropathy and renal neoplasia. The principal morphological feature of gasoline-induced nephropathy is accumulation of hyaline droplets (enlarged secondary lysosomes or phagolysosomes) in epithelial cells of the proximal convoluted tubule S1 and S2 segments. Inhibition of cathepsin B (a major lysosomal peptidase) by treatment of male rats with leupeptin causes rapid accumulation of phagolysosomes and alpha 2u-globulin in the kidney very similar to gasoline exposure. Further, the renal cortical subcellular distribution of alpha 2u-globulin, determined with an electron microscopic immunochemical method, is almost totally confined to phagolysosomes following administration of either gasoline or leupeptin. These results, taken together, indicate that the mechanism of nephrotoxicity of gasoline involves inhibition of renal phagolysosomal proteolysis.

A method for rapid, sensitive quantitation of short-patch DNA repair in cultured rat hepatocytes.

The sensitivity of DNA-repair detection after incubation of cultured rat hepatocytes with dimethylnitrosamine (DMN) and [Me-1',2'-3H]thymidine ([3H]TdR) was enhanced by the preparation of isolated nuclei. Nuclear isolation-liquid scintillation counting (NI-LSC) provided a rapid method for the determination of unscheduled DNA synthesis (UDS). DMN-induced UDS detected by autoradiography and NI-LSC correlated well and provided similar dose-response curves, indicating the utility of the NI-LSC method for the quantitation of short-patch DNA repair.

DNA strand breaks induced by hydrogen peroxide in isolated rat hepatocytes.

It has been proposed that increased rates of hepatic hydrogen peroxide (H2O2) production may initiate or promote the liver tumors that appear following chronic exposure of rodents to chemicals that cause peroxisome proliferation. However, the effect of H2O2 on the structural integrity of DNA in parenchymal hepatocytes, the target cells of peroxisome proliferator-induced carcinogenesis, is largely uncharacterized. Furthermore, oxidant-induced cellular damage has been invoked as causal of a number of hepatotoxic effects associated with exposure to chemicals other than peroxisome proliferators. For these reasons, alkaline elution analysis was used to study the action of H2O2, added exogenously, on DNA of intact, isolated rat hepatocytes. Addition of a bolus of H2O2 (0.01-1.0 mM) to monolayer cultures of hepatocytes caused concentration-dependent increases in single-strand DNA breaks (SSDB), which were maximal within 30 min of exposure. Cytotoxicity, as measured by lactate dehydrogenase (LDH) release, was minimal during a 1-h exposure to H2O2 concentrations less than 1 mM, but the efflux of oxidized glutathione was increased. Formation of SSDB was nearly linear with respect to H2O2 concentration in the range 0.1-1.0 mM. No double-strand DNA breaks or DNA-protein crosslinks were identified at H2O2 concentrations of 1 mM or less. Repair of SSDB in H2O2-free medium occurred in a rapid, linear manner only for the first 15 min, resulting in disappearance of 65% of the SSDB. A second, slower phase of SSDB rejoining occurred between 20 and 60 min of incubation in H2O2-free media; at 60 min rejoining was maximal (80% repair). These results define a specific type of DNA damage associated with H2O2 exposure of hepatocytes and suggest that primary cultures of rat hepatocytes are a suitable model for characterizing the potential genotoxic effects of oxidants, particularly excess H2O2 that may occur in the livers of animals exposed chronically to peroxisome proliferators.

Dominant role of cytochrome P-450 2E1 in human hepatic microsomal oxidation of the CFC-substitute 1,1,1,2-tetrafluoroethane.

The chlorofluorocarbon substitute 1,1,1,2-tetrafluoroethane (HFC-134a) is subject to metabolism by cytochrome P-450 in hepatic microsomes from rat, rabbit, and human. In rat and rabbit, the P-450 form 2E1 is a predominant low-KM, high-rate catalyst of HFC-134a biotransformation and is prominently involved in the metabolism of other tetrahaloalkanes of greater toxicity than HFC-134a [e.g. 1,2-dichloro-1,1-difluoroethane (HCFC-132b)]. In this study, we determined that the human ortholog of P-450 2E1 plays a role of similar importance in the metabolism of HFC-134a. In human hepatic microsomes from 12 individuals, preparations from subjects with relatively high P-450 2E1 levels were shown to metabolize HFC-134a at rates 5- to 10-fold greater than microsomes of individuals with lower levels of this enzyme; the increased rate of metabolism of HFC-134a was specifically linked to increased expression of P-450 2E1. The primary evidence for this conclusion is drawn from studies using mechanism-based inactivation of P-450 2E1 by diethyldithiocarbamate, competitive inhibition of HFC-134a oxidation by p-nitrophenol (a high-affinity substrate for P-450 2E1), strong positive correlation of rates of HFC-134a defluorination with p-nitrophenol hydroxylation in the study population, and correlation of P-450 2E1 levels with rates of halocarbon oxidation. Thus, our findings support the conclusion that human metabolism of HFC-134a is qualitatively similar to that of the species (rat and rabbit) used for toxicological assessment of this halocarbon.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitation of ketogenesis in periportal and pericentral regions of the liver lobule.

A method has been devised to quantitate rates of ketogenesis (acetoacetate + beta-hydroxybutyrate production) in discrete regions of the liver lobule based on changes in NADH fluorescence. In perfused livers from fasted rats, ketogenesis was inhibited nearly completely with either 2-bromoctanoate (600 microM) or 2-tetradecylglycidic acid (25 microM). During inhibition of ketogenesis, a linear relationship (r = 0.90) was observed between decreases in NADH fluorescence detected from the liver surface and decreases in ketone body production. NADH fluorescence was monitored subsequently from individual regions of the liver lobule by placing microlight guides on periportal and pericentral regions of the liver lobule visible on the liver surface. Rates of ketogenesis in sublobular regions were calculated from regional decreases in NADH fluorescence and changes in the rate of ketone body formation by the whole liver during infusion of inhibitors. In the presence of bromoctanoate, ketogenesis was reduced 80% and local rates of ketogenesis were decreased 31 +/- 4 mumol/g/h in periportal areas and 28 +/- 3 mumol/g/h in pericentral regions. Similar results were observed with tetradecylglycidic acid. Therefore, it was concluded that submaximal rates of ketogenesis from endogenous, mainly long-chain fatty acids are nearly equal in periportal and pericentral regions of the liver lobule in liver from fasted rats. Rates of ketogenesis and NADH fluorescence were strongly correlated during fatty acid infusion. Infusion of 250 microM oleate increased NADH fluorescence maximally by 8 +/- 1% over basal values in periportal regions and 17 +/- 4% in pericentral areas. Local rates of ketogenesis, calculated from these changes in fluorescence, increased 35 +/- 6 mumol/g/h in periportal areas and 55 +/- 5 mumol/g/h in pericentral regions. Thus, oleate stimulated ketogenesis nearly 60% more in pericentral than in periportal regions of the liver lobule.

Mechanism of zone-specific hepatic steatosis caused by valproate: inhibition of ketogenesis in periportal regions of the liver lobule.

Microvacuolar steatosis in periportal regions of the liver lobule was produced by injection of fasted rats with a single dose of valproate (500 mg/kg, subcutaneously). In livers perfused in the absence of exogenous fatty acids, ketone body (acetoacetate + beta-hydroxybutyrate) production was decreased by valproate (500 microM) maximally by 67%. Concomitantly, NADH fluorescence detected from the liver surface declined about 30% with a time course similar to that of the inhibition of ketogenesis. Valproate had little effect on oxygen uptake but caused an elevation of the steady state level of catalase-H2O2 corresponding to an increase in H2O2 production of about 6 mumol/g/hr. In addition, valproate decreased the rate of oxidized glutathione release into bile by 45% but had little effect on bile flow. In the presence of oleate (250 microM), valproate inhibited ketone body production by 46% and decreased NADH fluorescence by 39%. Rates of ketogenesis in periportal and pericentral regions of the liver lobule were calculated from changes in NADH fluorescence detected with micro-light guides during infusion of valproate in the presence and absence of fatty acids. In the absence of valproate, endogenous ketogenesis was about 35 mumol/g/hr in both regions of the liver lobule. In the presence of oleate, however, rates were significantly higher in pericentral regions (89 +/- 2 mumol/g/hr) than in periportal areas (71 +/- 3 mumol/g/hr). In the presence of added oleate, valproate decreased rates of ketogenesis to 34 +/- 4 mumol/g/hr in periportal regions and 51 +/- 3 mumol/g/hr in pericentral areas. We conclude, therefore, that fat accumulates in periportal areas because valproate depresses ketogenesis to a greater extent in hepatocytes localized around the portal triad.

Modulation of glucose metabolism in isolated rat hepatocytes by 1,1,1,2-tetrafluoroethane.

The thermodynamic behavior and lack of ozone-depleting potential of 1,1,1,2-tetrafluoroethane (R-134a) suggest it as a likely replacement for dichlorodifluoromethane (R-12), now used as the refrigerant in many air-conditioning systems. To further the presently incomplete toxicological analysis of R-134a, the effects of R-134a on cell viability and functional competence of glucose metabolism were evaluated in suspension cultures of hepatocytes derived from fed or fasted rats. R-134a concentrations up to and including 75% (750,000 ppm) in the gas phase of sealed culture flasks did not produce evidence of cytolethality (LDH leakage) following 2 hr of exposure; in contrast, halothane (1,1,1-trifluoro-2-bromo-2-chloroethane) caused cell death at a gas phase concentration of only 1250 ppm. In hepatocytes isolated from fed rats. R-134a at concentrations of 12.5 to 75% increased glycolysis (production of lactate + pyruvate) in a concentration-dependent manner; no effect was observed at 5%. At 25%, R-12 and 1,1,2,2-tetrafluoro-1,2-dichloroethane (R-114) were of equal potency to R-134a in stimulating glycolysis: 1,1,1,2,2-pentafluoro-2-chloroethane (R-115) depressed glycolysis slightly. Halothane, at concentrations as low as 300 ppm, markedly increased rates of glycolysis. Glucose production by hepatocytes of fed rats was decreased by R-134, R-12, and R-114 only at concentrations of 25% or more. On the other hand, halothane (greater than or equal to 300 ppm) potently decreased glucose production by hepatocytes. In cells isolated from livers of fasted rats, R-134a exposure inhibited gluconeogenesis in a concentration-dependent manner although this effect was not significant until R-134a concentrations reached 12.5%. Comparative potency studies showed that R-134a, R-12, or R-114 (25% gas phase) inhibited gluconeogenesis about equally while as little as 300 ppm halothane was effective and R-115 (25%) was without effect. Considering that the threshold for alteration of the rate of glucose metabolism in this in vitro paradigm is about 12.5% R-134a, we conclude that toxicologically significant alteration of glucose-linked bioenergetics is unlikely at the levels of R-134a exposure anticipated in workplace or environment.

Defluorination of 1,1,1,2-tetrafluoroethane (R-134a) by rat hepatocytes.

As part of its toxicological evaluation we assessed the in vitro metabolism of 1,1,1,2-tetrafluoroethane (R-134a), a non-ozone-depleting chemical likely to replace dichlorodifluoromethane (R-12) as an air-conditioning refrigerant. Hepatocyte suspensions in sealed flasks produced increasing quantities of F- (detected in the liquid media) as the headspace concentration of R-134a increased from 1% to 50% (balance of atmosphere 95% O2-5% CO2); the kinetics of defluorination suggested substrate-saturation. Little F- was detected in cultures without R-134a or in cell suspensions heated prior to addition of R-134a. Halothane (1,1,1-trichloro-2-bromo-2-chloro-ethane), although not defluorinated by hepatocytes maintained with 95% O2, inhibited defluorination of R-134a. Hepatocytes from phenobarbital-treated rats dehalogenated high (greater than or equal to 25%) concentrations of R-134a at greater rates than cells from untreated rats. These findings are consistent with the hypothesis that oxidative metabolism of R-134a by cytochrome P-450 can occur in vivo.

A comparison of male rat and human urinary proteins: implications for human resistance to hyaline droplet nephropathy.

alpha 2u-Globulin (alpha G), the major urinary protein of sexually mature male rats, is a key determinant of susceptibility to hyaline droplet nephropathy (HDN) induced by a variety of hydrocarbons in male rats. Arguments against extrapolating renal toxicity and carcinogenicity data for HDN-inducing toxicants from male rats to risk assessment for humans rely on the observation that humans do not express alpha G. Yet, human serum and urine are known to contain proteins coded for by the same gene family that also controls alpha G synthesis in the rat. Therefore, to understand some of the quantitative and qualitative differences between proteins of human and male rat urine which confer apparent resistance to HDN in humans, urinary proteins of male F344 rats (ca. 3 months old) and normal human males were compared by cation exchange, gel filtration, SDS-PAGE, and partially identified by Western blotting. We observed that (1) the protein content of human urine is only 1% that of male rat urine; (2) human urinary proteins, recovered by (NH4)2SO4 precipitation followed by dialysis, are primarily of high (greater than or equal to 75 kDa) molecular weight (MW) with minor components of 12-66 kDa; (3) male rat urine has little high-MW protein, but is rich in alpha G (18.5 kDa); (4) at pH 5, the most cationic fraction of human urinary protein constituted only about 4% of the total while the analogous fraction of rat urine, containing alpha G, contained 26% of total urinary protein; and (5) cationic (at pH 5.0) human urinary proteins included small amounts of proteins, e.g., alpha 1-acid glycoprotein, and alpha 1-microglobulin, which are products of the gene family coding for alpha G in rat. Thus, although humans excrete trace amounts of proteins similar to alpha G, the very low protein content of human urine, the relatively small proportion of cationic to total proteins, and the high MW of the most abundant human urinary proteins form a biological basis for suggesting that humans are not at risk for the type of fuel and solvent hydrocarbon-induced nephropathy, and the sequelae of such nephropathy, observed in male rats.

Metabolism in vivo and in vitro of the refrigerant substitute 1,1,1,2-tetrafluoro-2-chloroethane.

Ternary mixtures of hydrochlorofluorocarbons and hydrofluorocarbons are being evaluated as refrigerant substitutes for dichlorodifluoromethane, which is to be banned from further production in 2000. A priori consideration of the similarity between 1,1,1,2-tetrafluoro-2-chloroethane (HCFC-124), a primary component of candidate refrigerant blends, and halothane suggests that metabolism of HCFC-124 might proceed via reactive intermediates. Our data show that rats exposed for 2 hr to approximately 10,000 ppm HCFC-124 excreted both inorganic fluoride (F-) and trifluoroacetic acid (TFA), identified by 9F-NMR, in the urine. Likewise, microsomes produced F- and TFA from HCFC-124 in an NADPH-dependent, CO-inhibited, aerobic reaction. Treatment of rats with pyridine caused about a 20-fold increase in aerobic microsomal metabolism (F- release) of HCFC-124, while the rate of defluorination was slightly decreased by phenobarbital administration. An antibody to cytochrome P450 IIE1 inhibited more than 90% of HCFC-124 metabolism in pyridine-induced preparations. Defluorination of HCFC-124 by microsomes also occurred under conditions of greatly reduced oxygen tension, demonstrating that this halocarbon can be reductively metabolized. Moreover, heat-inactivated, NADPH-reduced microsomes liberated F- and a fluorinated organic product, although not TFA, from HCFC-124. Formation of TFA and F- as products of oxidative HCFC-124 metabolism support the hypothesis that trifluoroacetyl fluoride is formed as an intermediate. Trifluoroacetyl halides are known to adduct tissue proteins. The reductive metabolism of HCFC-124, by analogy to halothane, may produce a radical (CHFCF3) capable of biological interactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Accumulation of alpha 2u-globulin in the renal proximal tubules of male rats exposed to unleaded gasoline.

Saturated branched-chain aliphatic hydrocarbons, found in motor fuels, induce nephrotoxicity in male rats. Treatment of male rats with unleaded gasoline (0.04-2.0 ml/kg body wt, po) for 9 days increased markedly the number and size of hyaline (protein resorption) droplets in epithelial cells of the renal proximal convoluted tubules (PCT) and enhanced cellular exfoliation at high dose levels. No other treatment-related pathological effects were observed in the glomeruli, distal tubules, or medulla. The renal content of alpha 2u-globulin, a major urinary protein of male rats, was increased maximally by about 4.4-fold after gasoline administration (1.0 ml/kg, po, 9 days); no further increase was observed at higher doses. Immunoperoxidase staining of kidney tissue sections for alpha 2u-globulin revealed large accumulations of antigen localized in many of the PCT epithelial cells which contained hyaline droplets. The hepatic content of alpha 2u-globulin and its mRNA were not altered by gasoline administration. These data show, for the first time, that alpha 2u-globulin is accumulated in the kidneys of gasoline-intoxicated male rats and sequestered specifically in some of the hyaline droplets characteristic of gasoline-induced nephropathy. A hydrocarbon-induced defect in the renal lysosomal degradation of low-molecular-weight urinary proteins, rather than increased synthesis of these proteins, appears to cause hyaline droplet accumulation.

Establishing guidance for the handling and containment of new chemical entities and chemical intermediates in the pharmaceutical industry.

This information is intended to guide scientists and technicians working with pharmaceutical substances early in development, before occupational exposure levels (OELs) can be set. The focus is on determining hazard categories, or occupational exposure bands, which may be applied temporarily until full health-based OEL's are in place.

Hydrocarbon-induced hyaline droplet nephropathy in male rats during senescence.

Male rats administered unleaded gasoline rapidly develop nephropathy characterized by accumulation of hyaline droplets in cells of the proximal convoluted tubules (PCT). This acute response is implicated in development of renal carcinoma in male rats exposed chronically to wholly volatilized gasoline. A major constituent of hyaline droplets is alpha 2 mu-globulin, a protein of hepatic origin for which the rate of synthesis declines during aging. Little information, however, is presently available on possible age-dependent susceptibility of male rats to hydrocarbon-induced nephropathy. In kidneys of untreated male Fischer 344 rats the number of constitutive hyaline droplets declined progressively with increasing age. Electrophoresis of renal cortical homogenates revealed a protein with Mr about 18 X 10(3), probably alpha 2 mu-globulin, in young (3.5 months old) male rats and total absence of this protein in aged (26 months old) males. RIA confirmed that constitutive levels of renal and hepatic alpha 2 mu-globulin in old rats were less than 1.5% of those in young adults. Unleaded gasoline (0.4 ml/kg/day, po, 5 days) caused accumulation of hyaline droplets in renal PCT of 3.5-month-old males accompanied by a marked increase (about twofold) in the renal content of alpha 2 mu-globulin, whereas the same treatment was without effect in 26-month-old rats. Finally, in the renal cortex of young rats the activities of the lysosomal proteases cathepsin B and D were increased following gasoline administration, presumably in response to protein accumulation. However, in 26-month-old rats cathepsin B activity was unaffected, while cathepsin D was increased by gasoline administration. Thus, we conclude that animal age is an important determinant in the development of hydrocarbon-induced nephropathy and only rats which produce large amounts of alpha 2 mu-globulin are susceptible to development of this pathology.

Cloning and expression of the rat liver cDNA for peroxisomal enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase in lambda GT11. Transcriptional regulation of enzyme activity by Wy-14643 in primary cultures of rat hepatocytes.

Proliferation of rat liver peroxisomes by the hypolipidemic drug Wy-14643 is associated with a concomitant induction of peroxisomal enzymes involved in the beta-oxidation of fatty acids. In order to explore the molecular mechanism of this induction process we have cloned the cDNA for the peroxisomal bifunctional enzyme enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase (ECH) in the lambda gt11 expression vector. The library was screened with the monospecific rabbit antiserum to ECH. Hybrid-selected-mRNA translation established that the immunoreactive clones contain the cDNA sequences of the ECH bifunctional enzyme. The cloned cDNA was used to define the early events associated with enzyme induction in primary cultures of rat hepatocytes. Dot-blot hybridization of the total hepatocyte RNA with the ECH cDNA probe showed that the ECH mRNA begins to rise at about 10-15 h following incubation with Wy-14643. At 24 h and 48 h of incubation the stimulation of the ECH mRNA over the vehicle-treated control reached 26-fold and 47-fold respectively. Run-off experiments in the isolated nuclei of hepatocytes showed no increase in the transcription rate of the ECH gene at 5 h after drug treatment and a 2-fold and 11-fold increase at 10 h and 20 h of drug treatment. From these results we conclude that the increase in ECH activity by Wy-14643 is due to an enhancement of the rate of transcription of the ECH gene. However, the relatively long lag period of about 10-15 h after exposure of hepatocytes to Wy-14643 suggests that the induction of the ECH mRNA may involve an indirect effect of the drug on the transcription of this gene.

Rapid postexposure decay of alpha 2u-globulin and hyaline droplets in the kidneys of gasoline-treated male rats.

Unleaded gasoline induces nephropathy, characterized by rapid accumulation of hyaline (protein resorption) droplets in epithelial cells of the renal proximal convoluted tubules, only in male rats. The hepatic synthesis of the male rat-specific protein alpha 2u-globulin, a constituent of renal hyaline droplets, is unaltered by gasoline treatment (Olson et al., 1987). Renal alpha 2u-globulin content increased to 210% of control within 18 h of a single oral dose of gasoline (2.0 ml/kg); maximal levels (320% of control) were attained following gasoline administration for 3 d. Increases in renal alpha 2u-globulin caused by gasoline were accompanied by concurrent proliferation of hyaline droplets. However, within 3 d of terminating gasoline administration renal alpha 2u-globulin content decreased to the same level as that in unexposed rats, although renal hyaline droplet number returned to pretreatment levels somewhat more slowly. The conjoint effect of postexposure recovery and estradiol (an inhibitor of hepatic alpha 2u-globulin synthesis) administration was also determined in male rats. On postexposure d 3, 6, and 9, estradiol treatment (1 mg/kg, sc, 4 d, starting on d 9 of gasoline treatment) decreased renal alpha 2u-globulin content to 75%, 59%, and 48%, respectively, of that in rats allowed to recover from gasoline with no hormone treatment. Hepatic alpha 2u-globulin content in estradiol-treated rats was decreased by 74%, 97%, and 96% at the same intervals. Estradiol treatment during recovery from gasoline also appeared to increase the removal of accumulated hyaline droplets from the renal cortex. Thus, accumulation of alpha 2u-globulin-containing hyaline droplets after subacute exposure of male rats to gasoline is rapidly reversible, dependent on continuous exposure to gasoline and maintenance of the normal rate of hepatic alpha 2u-globulin synthesis. These results emphasize the dynamic state of renal cortical hyaline droplets and suggest strongly that gasoline hydrocarbons cause hyaline droplet accumulation by prolonging the half-time of degradation of alpha 2u-globulin.

Oxidative defluorination of 1,1,1,2-tetrafluoroethane by rat liver microsomes.

1,1,1,2-Tetrafluoroethane (R-134a) is a non-ozone-depleting alternative to dichlorodifluoromethane for use as an air-conditioning refrigerant and as a propellant in anti-asthmatic and other pharmaceutical preparations. Hepatic microsomes, supplemented with NADPH, catalyzed the release of F- from R-134a; metabolite production was positively correlated with both duration of incubation and gas phase [R-134a]. Defluorination of R-134a was inhibited by CO, lack of NADPH, or heat denaturation of microsomes. Release of F- from R- 134a biotransformation as shown by the near-total lack of dehalogenation during anaerobic incubations. R-134a did not produce a difference spectrum (360 to 500 nm) with either oxidized or dithionite-reduced microsomes. Microsomes from phenobarbital- or Aroclor 1254-treated rats produced greater amounts of F- per mg protein from high concentrations of R-134a than did microsomes from untreated rats, but when normalized for microsomal cytochrome P-450 content both phenobarbital and Aroclor treatment decreased the specific activity (nmol F-/nmol cytochrome P-450) of R-134a metabolism. Furthermore, while defluorination of R-134a by microsomes from livers of untreated rats was substrate-saturable (Vmax, 11 nmol of F-/nmol cytochrome P-450/15 min; KM, 8% R-134a), R-134a dehalogenation by microsomes from Aroclor-treated rats was nonsaturable with [R-134a] as high as 69%. Microsomes from phenobarbital-treated rats retained the saturable, low KM activity, but also exhibited the apparently nonsaturable kinetic component when [R-134a] was greater than 24%.(ABSTRACT TRUNCATED AT 250 WORDS)