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.

Isoflurane-chlorodifluoroethene interaction in human liver microsomes. Role of cytochrome P4502B6 in potentiation of haloethene metabolism.

Short-chain saturated halocarbons, including isoflurane and the chlorofluorocarbon substitute HCFC-123, can strongly potentiate the cytochrome P450-dependent oxidation of gaseous haloethenes, such as 2-chloro-1,1-difluoroethene (CDE) and vinyl chloride, in vivo and in vitro. P450 isozyme specificity in this effect is suggested by the fact that the interaction is pronounced in microsomes from rats treated with phenobarbital, but does not occur in microsomes of isoniazid- or beta-naphthoflavone-treated animals. We examined the effect of isoflurane on CDE defluorination in liver microsomes from 10 human organ donors to determine whether saturated halocarbon/haloethene interactions also occur in humans and, if so, to determine the cytochromes P450 involved. Three of the samples exhibited isoflurane-stimulated increases (24, 32, and 41%) in CDE defluorination; isoflurane either inhibited or had no effect on CDE metabolism in the other seven samples. Two samples in which isoflurane potentiated CDE metabolism to the greatest rates had higher coumarin 7-hydroxylase (indicative of CYP2A6), 7-ethoxycoumarin O-deethylase (CYP2B6), and nifedipine oxidase (CYP3A4) activities than the other eight samples. However, all 10 subjects had similar rates of phenacetin O-deethylation (CYP1A2) and chlorzoxazone 6-hydroxylation (CYP2E1). In microsomes from cells transfected with cDNAs coding for individual human P450s, CDE metabolism by CYP2B6 was stimulated (216%) by isoflurane, whereas isoflurane did not stimulate CDE metabolism by human CYP2A6, CYP3A4, CYP2D6, or CYP2E1. Isoflurane highly increased CDE defluorination in purified rat CYP2B1 (470%).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Pentahaloethane-based chlorofluorocarbon substitutes and halothane: correlation of in vivo hepatic protein trifluoroacetylation and urinary trifluoroacetic acid excretion with calculated enthalpies of activation.

The hydrochlorofluorocarbons (HCFCs) 2,2-dichloro-1,1,1-trifluoroethane (HCFC-123) and 2-chloro-1,1,1,2-tetrafluoroethane (HCFC-124) and the hydrofluorocarbon (HFC) pentafluoroethane (HFC-125) are being developed as substitutes for chlorofluorocarbons that deplete stratospheric ozone. The structural similarity of these HCFCs and HFCs to halothane, which is hepatotoxic under certain circumstances, indicates that the metabolism and cellular interactions of HCFCs and HFCs must be explored. In a previous study [Harris et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1407], similar patterns of trifluoroacetylated proteins (TFA-proteins) were detected by immunoblotting with anti-TFA-protein antibodies in livers of rats exposed to halothane or HCFC-123. The present study extends these results and demonstrates that in vivo TFA-protein formation resulting from a 6-h exposure to a 1% atmosphere of these compounds follows the trend: halothane approximately HCFC-123 much greater than HFC-124, greater than HFC-125. The calculated enthalpies of activation of halothane, HCFC-123, HCFC-124, and HFC-125 paralleled the observed rate of trifluoroacetic acid excretion in HCFC- or HFC-exposed rats. Exposure of rats to a range of HCFC-123 concentrations indicated that TFA-protein formation was saturated at an exposure concentration between 0.01% and 0.1% HCFC-123. Deuteration of HCFC-123 decreased TFA-protein formation in vivo. Urinary trifluoroacetic acid excretion by treated rats correlated with the levels of TFA-proteins found after each of these treatments. No TFA-proteins were detected in hepatic fractions from rats given 1,1,1,2-tetrafluoroethane (HFC-134a), which is not metabolized to a trifluoroacetyl halide.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

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.

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)

Oxidation of 1,1,1,2-tetrafluoroethane in rat liver microsomes is catalyzed primarily by cytochrome P-450IIE1.

1,1,1,2-Tetrafluoroethane (R-134a), a nonozone-depleting alternative air-conditioning refrigerant and propellant for pharmaceutical preparations, is oxidatively defluorinated by rat hepatic microsomes. In this report we show that induction of cytochrome P-450IIE1 in rats, by pyridine administration, resulted in an 8-fold increase in the rate of R-134a metabolism by hepatic microsomes (Vmax 47 vs. 6 nmol F-/mg microsomal protein/15 min). Furthermore, when data were normalized for P-450 content, a 4-fold increase in R-134a metabolism was noted for IIE1-enriched microsome preparations. In contrast, phenobarbital and Aroclor 1254 decreased the specific activity of hepatic microsomes for this function. The microsomal content of P-450IIE1, as evaluated by Western blot, was elevated significantly only in microsomes from pyridine-treated rats. p-Nitrophenol and aniline, which are metabolized at high rates by rat P-450IIE1, decreased the rate of R-134a defluorination by hepatic microsomes; Dixon plot analysis indicated competitive inhibition with a Ki of 36 microM p-nitrophenol or 115 microM aniline. Pyridine also potently induced defluorination of R-134a catalyzed by rabbit liver microsomes. Studies with individual P-450 isozymes purified from rabbit liver showed that the phenobarbital- and polycyclic hydrocarbon-induced isozymes (IIB1 and IA2) defluorinated R-134a at negligible rates (1.9 and 0.4 nmol F-/nmol P-450/60 min, respectively). In contrast, P-450IIE1 catalyzed defluorination of R-134a at a relatively high rate (16.2 nmol F-/nmol P-450/60 min); isozyme IA1, which also is induced by nitrogen-containing heterocycles such as pyridine, was somewhat active (5.3 nmol F-/nmol P-450/60 min).(ABSTRACT TRUNCATED AT 250 WORDS)

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)

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.

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.

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.

Relationship of oxidative damage to the hepatocarcinogenicity of the peroxisome proliferators di(2-ethylhexyl)phthalate and Wy-14,643.

Quantitative comparisons of the time course of biochemical and morphological changes induced by peroxisome proliferators resulting in low and high incidences of hepatic cancer have not been conducted previously under bioassay conditions. [4-Chloro-6-(2,3-xylidino)-2-pyrimidyl-thio]acetic acid (Wy-14,643) at 0.1% in the diet produced a much higher incidence of hepatic cancer in male rats than 1.2% di(2-ethylhexyl)phthalate (DEHP) in the diet. Both diets, however, caused similar degrees of peroxisome proliferation. To investigate this difference in carcinogenicity, H2O2-detoxification mechanisms and indices of oxidative damage were evaluated in male F-344 rats fed 1.2% DEHP or 0.1% Wy-14,643 for up to one year. DEHP or Wy-14,643 treatment increased hepatic catalase activity approximately 25% from 8 to 365 days. DEHP or Wy-14,643 treatment decreased hepatic glutathione peroxidase activity by 50% from 8 to 365 days. Glutathione concentrations were not affected by 151 days of DEHP or Wy-14,643 feeding. The similar effects of DEHP and Wy on H2O2 detoxification enzymes and glutathione concentrations suggests that these factors are not responsible for the widely different carcinogenicities of Wy-14,643 and DEHP. Hepatic vitamin E concentrations were 50% lower in rats receiving Wy-14,643 for 151 days as compared to rats fed DEHP or control diets. Lipofuscin, which was contained within lysosomes, was increased 3-fold after 39 days of DEHP and remained at this level up to 365 days of treatment. In comparison, lipofuscin was increased 4-fold after 18 days of Wy-14,643 and continued to accumulate in a linear manner reaching values 30-fold over controls after 365 days of treatment. DEHP treatment for 39-365 days increased the activities of the lysosomal enzymes alpha-fucosidase, beta-galactosidase and N-acetylglucosaminidase 50-100%. The same enzyme activities were increased approximately 4-fold after 39-365 days of Wy-14,643. Lysosomal cathepsin B activity was unchanged by DEHP but doubled by 151 and 365 days of Wy-14,643. Acid phosphatase activity was unchanged by DEHP but increased by 50% after 151 and 365 days of Wy-14,643. In addition, conjugated dienes were increased (approximately 45%) only in rats receiving Wy-14,643 for 151 and 365 days. These data show for the first time that the magnitude and time course of lipofuscin deposition, induction of lysosomal enzymes and conjugated diene accumulation, is correlated closely with the degree of carcinogenicity. Wy-14,643-induced decreases in hepatic vitamin E concentrations could contribute to the observed accumulation of conjugated dienes at later time points.(ABSTRACT TRUNCATED AT 400 WORDS)

Phagolysosomal alterations induced by unleaded gasoline in epithelial cells of the proximal convoluted tubules of male rats: effect of dose and treatment duration.

Short-term oral administration of unleaded gasoline to male rats reproduces the accumulation of phagolysosomes (hyaline droplets) in epithelial cells of the renal proximal convoluted tubules (PCT) observed following long-term inhalation of wholly volatilized gasoline. Phagolysosomes are partially composed of alpha 2u-globulin, a low-molecular-weight protein, unique to male rats. In this study, dose-dependent and chronologic alterations of phagolysosomes caused by gasoline were observed by transmission electron microscopy. Exposure to commercially available unleaded gasoline (0.4-2.0 ml/kg, po, once daily, 9 d) increased the number and size of phagolysosomes in epithelial cells of the PCT in male rat kidney. However, administration of 0.04 ml gasoline/kg or less was ineffective in inducing phagolysosomal accumulation. Subcellular analysis revealed that many of the phagolysosomes observed in treated rats (doses greater than 0.4 ml/kg) were angular and had cross-sectional diameters varying from 0.5 to 9 microns; in controls the majority of phagolysosomes were round and their diameter varied from 0.5 to 2.5 microns. Treatment of male rats with gasoline (2.0 ml/kg body weight, po, 1-9 d) caused a progressive increase in the number and size of phagolysosomes in PCT epithelial cells dependent on treatment duration. Alterations in phagolysosomal morphology and quantity occurred within 20 h following a single dose of gasoline, emphasizing that the process of phagolysosome accumulation is a dynamic phenomenon. Many of the enlarged phagolysosomes contained a condensed, crystalline core of greater electron density than the surrounding matrix. Furthermore, the rapid increase in abnormal, condensed contents in the phagolysosomes may indicate that a derangement of renal protein catabolism is the primary mechanism by which fuel hydrocarbons cause hyaline droplet nephropathy in male rats.

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.

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.

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.