Mechanism of chloroform nephrotoxicity : I. Time course of chloroform toxicity in male and female mice

Chloroform (CHCl3) nephrotoxicity in male mice could be detected as early as 2 hr after CHCl3 administration (250 microliter/kg, sc) as decreased ability of renal cortical slices to accumulate p-aminohippurate (PAH) and tetraethylammonium (TEA). The decrease was preceded and paralleled by a reduction of renal cortical nonprotein sulfhydryl (NPSH) concentration, an index of tissue reduced glutathione concentration. Histologic alterations were not observed until NPSH concentrations and PAH and TEA accumulation had reached the nadir, 5 hr after CHCl3 administration. Female mice exhibited no evidence of nephrotoxicity to CHCl3 even when the dose was increased to 1000 microliter/kg or when pretreated with diethyl maleate to reduce renal cortical NPSH concentrations prior to CHCl3 injection. The extent of hepatotoxicity was similar in male and female mice and decreases of hepatic NPSH concentrations also were detected by 1.5 hr after CHCl3 administration. The rapid response of the kidney to CHCl3 toxicity in male mice and the similarity of liver toxicity in both sexes suggests that nephrotoxicity occurs independently of hepatotoxicity. Furthermore, the ability to detect these early changes in vivo following CHCl3 administration may permit the development of an in vitro model to evaluate the mechanism of CHCl3 nephrotoxicity.     

Mechanism of chloroform nephrotoxicity: IV. Phenobarbital potentiation of in vitro chloroform metabolism and toxicity in rabbit kidneys

Metabolism of chloroform (CHCl₃) by a cytochrome P-450-dependent process to a reactive metabolite may be required to elicit hepatic and renal toxicities. Specific inducers or inhibitors of cytochrome P-450 have been employed frequently as tools to demonstrate this relationship between metabolism and toxicity in the liver. The experiments reported herein were designed to identify the relationship between metabolism and toxicity of CHCl₃ in the kidney of rabbits, a species in which renal cytochrome P-450 is induced by phenobarbital. Pretreatment with phenobarbital enhanced the toxic response of renal cortical slices to CHCl₃in vitro as indicated by decreased p-aminohippurate and tetraethylammonium accumulation. Phenobarbital pretreatment also potentiated in vitro¹⁴CHCl₃ metabolism to ¹⁴CO₂ and covalently bound radioactivity in rabbit renal cortical slices and microsomes. Addition of l-cysteine significantly reduced covalent binding in renal microsomes from both phenobarbital-treated and control rabbits and was associated with the formation of the radioactive phosgene-cysteine conjugate 2-oxothiazolidine-4-carboxylic acid (OTZ). Formation of OTZ was enhanced in renal microsomes from phenobarbital-pretreated rabbits. Thus, this in vitro model supports the hypothesis that the kidney metabolizes CHCl₃ to the nephrotoxic metabolite, phosgene.     

Mechanism of chloroform nephrotoxicity : II. In vitro evidence for renal metabolism of chloroform in mice

Preincubation of renal cortical slices with chloroform (CHCl₃) from male, but not female, mice resulted in a subsequent decrease of the ability of the slices to accumulate the organic ions, p-aminohippurate (PAH) and tetraethylammonium (TEA). These sex-related differences, the time required for manifestation of this effect (60 to 90 min), and the concentration dependency (0 to 50 μmol, 0 to 4 μl CHCl₃) were similar to in vivo observations on CHCl₃ nephrotoxicity in mice. Furthermore, an equimolar concentration of deuterated CHCl₃ (CDCl₃) in vitro was less effective than CHCl₃ in decreasing PAH and TEA accumulation in male renal cortical slices. The effects of CHCl₃ on PAH and TEA accumulation could be diminished or blocked by preincubation with CHCl₃ in the presence of carbon monoxide or at 0°C, respectively. The nephrotoxicity of CHCl₃ in vitro was increased in renal cortical slices from male mice pretreated with diethyl maleate. Thus, this in vitro model with mouse renal cortical slices and the sex-related differences in CHCl₃ nephrotoxicity suggests that the kidney may metabolize CHCl₃ in situ to a nephrotoxic metabolite.     

Mechanism of chloroform nephrotoxicity. III. Renal and hepatic microsomal metabolism of chloroform in mice

In vitro studies with male ICR mouse renal cortical slices have indicated that chloroform (CHCl3) is metabolized by the kidney to a nephrotoxic intermediate, possibly by a cytochrome P-450-dependent mechanism similar to that occurring in the liver. In this investigation, metabolism of 14CHCl3 by microsomes prepared from renal cortex and liver provided definitive evidence for a role of cytochrome P-450 in the renal metabolism and toxicity of CHCl3. 14CHCl3 was metabolized to 14CO2 and covalently bound radioactivity by male renal cortical microsomes; metabolism required oxygen, a NADPH regenerating system, was dependent on incubation time, microsomal protein concentration, and substrate concentration, and was inhibited by carbon monoxide. Consistent with the absence of CHCl3 nephrotoxicity in female mice, little or no metabolism of 14CHCl3 by female renal cortical microsomes was detected. CHCl3 produced a type I binding spectrum with oxidized male renal cortical and hepatic microsomes. Incubation of glutathione with microsomes and 14CHCl3 increased the amount of aqueous soluble metabolites detected with a concomitant decrease of metabolism to 14CO2 and covalently bound radioactivity, suggesting the formation of a phosgene conjugate as has been described for hepatic CHCl3 metabolism. These data support the hypothesis that renal cytochrome P-450 metabolizes CHCl3 to a nephrotoxic intermediate.     

Identification and quantification of urinary metabolites of dinitrotoluenes in occupationally exposed humans

Rats exposed to technical grade dinitrotoluene (DNT) develop hepatocellular carcinomas. Humans may be exposed to DNT during its manufacture and use. To permit comparisons of human excretion patterns of DNT metabolites with those previously observed in rats, urine specimens were collected over a 72-hr period from workers at a DNT manufacturing plant. Samples were analyzed for 2,4- and 2,6-DNT and putative metabolites by gas chromatography-mass spectrometry. Urine from workers exposed to DNT contained 2,4- and 2,6-DNT, 2,4- and 2,6-dinitrobenzoic acid, 2,4- and 2,6-dinitrobenzyl glucuronide, 2-amino-4-nitrobenzoic acid, and 2-(N-acetyl)amino-4-nitrobenzoic acid. Excretion of these metabolites peaked near the end of the workshift, but declined to either very low or undetectable concentrations by the start of work the following day. The calculated half-times for elimination of total DNT-related material detected in urine ranged from 1.0 to 2.7 hr, and those of individual metabolites from 0.8 to 4.5 hr. The most abundant metabolites were 2,4-dinitrobenzoic acid and 2-amino-4-nitrobenzoic acid, collectively accounting for 74 to 86% of the DNT metabolites detected. The data indicate that urinary metabolites of DNT in humans are qualitatively similar to those found in rats, but quantitative differences exist in the relative amounts of each metabolite excreted.     

Influence of simultaneous exposure to acrylonitrile and styrene on the toxicity and metabolism of styrene in rats

Nine groups of adult male rats were given different combinations of styrene and acrylonitrile and each chemical was administered at three doses (styrene 0, 5.8, and 11.6 mmol/kg, ip; acrylonitrile 0, 0.3, and 0.6 mmol/kg, po). The animals were killed 24 hr later and blood and urine samples were collected. The results of biochemical analyses due to the toxicity of both chemicals and of the determination of urinary metabolites of styrene were then subjected to a factorial (3 X 3) analysis of variance. There was: (1) a significant elevation of blood urea nitrogen (BUN) and serum glutamic-pyruvic transaminase (SGPT), and a diminution of urinary creatinine due to styrene; (2) an increase in serum creatinine and serum glutamicoxaloacetic transaminase (SGOT) due to styrene that was further increased by acrylonitrile; and (3) an increase in the concentrations of urinary metabolites (thioethers, mandelic, phenylglyoxylic, and hippuric acids) due to styrene that was considerably reduced by acrylonitrile. These results suggest that styrene causes renal toxicity which may be potentiated by acrylonitrile; furthermore, the significant diminution of the urinary metabolites of styrene due to acrylonitrile obscures interpretation of the results of the biological monitoring of exposure to styrene.     

Nicotyrine inhibits in vivo metabolism of nicotine without increasing its toxicity

The effect of pretreatment with β-nicotyrine on tissue concentrations of nicotine and metabolically formed cotinine was studied in male albino mice injected with [¹⁴C]nicotine. Acute toxicity of nicotine administered ip or iv was also compared in the pretreated and untreated animals. The pretreatment with nicotyrine resulted in a significant dose-dependent increase of nicotine concentrations in the liver, blood, and brain. Concomitantly, there was a significant decrease in the cotinine concentrations suggesting an inhibition of nicotine liver metabolism. Despite the higher concentrations of nicotine in the brain of the pretreated mice, the LD50 after an ip injection of nicotine was not different from the untreated animals. On the other hand there was complete protection against the lethal effect of iv-injected nicotine in the pretreated animals suggesting a direct protective interaction of nicotyrine with nicotine in CNS.     

Influence of strain differences in mice on the metabolism and toxicity of benzene

Chronic benzene exposure results in a progressive depression of bone marrow function and is thought to be caused by a metabolite of benzene (Snyder and Kocsis, 1975; Goldstein and Laskin, 1977). Several reports concerning differences in xenobiotic metabolism and toxicity among inbred strains of mice prompted us to study benzene metabolism and toxicity in C57BL/6 and DBA/2 mice. DBA/2 mice were more susceptible to benzene than C57BL/6 mice. No differences in the total amount of urinary benzene metabolites produced were found between the strains; however, differences in the relative amounts of specific metabolites were noted. DBA/2 mice produced more phenylglucuronide but less ethereal sulfate conjugates than C57BL/6 mice. Hydrolysis of the urinary conjugates revealed that DBA/2 mice excreted more phenol, but less hydroquinone than C57BL/6 mice. Multiple dose studies revealed that the more resistant C57BL/6 mice contained less water soluble benzene metabolites in bone marrow, liver, kidney, blood, spleen, and lung than DBA/2 mice. C57BL/6 mice also contained less covalently bound metabolites in bone marrow, blood, spleen, and muscle than DBA/2 mice following multiple doses of benzene. V_max values for UDPGA utilization in C57BL/6 mice were almost six times the V_max values observed for DBA/2 mice. Furthermore, V_max values for phenylsulfate formation in C57BL/6 mice were three times the V_max values for DBA/2 mice. It was concluded that the difference in susceptibility to benzene between C57BL/6 and DBA/2 mice was not the result of a single factor, buth rather, the sum total of a number of metabolic events.     

Influence of dietary rapeseed oil and erucic acid upon myocardial performance and hemodynamics in rats

Rats fed rapeseed oil, pure erucic acid, or a control diet of sunflowerseed oil during 24-26 weeks were studied for effects upon mechanical behavior of the isolated perfused heart and upon myocardial performance and hemodynamics in intact animals both under basal and stimulated conditions. In spite of focal myocardial fibrotic lesions due to rapeseed oil, no changes were found with respect to the intrinsic myocardial contractility in vitro and and in vivo. After inotropic intervention, only the rapeseed oil fed animals showed less contractile reserve capacity. The absence of this effect in the erucic acid-treated animals is in agreement with the histological studies showing no epicardiac fibrotic lesions in these animals. It appears that erucic acid is able to interfere with the contractile system of the peripheral vascular system. Both in the rapeseed oil-treated group and the erucic acid-treated group, the vasoconstrictor response toward norepinephrine was profoundly reduced. In all three oil fed groups, isoproterenol reduced myocardial contractility which has been attributed to a lowered perfusion pressure in the coronary blood supply of the myocardium with simultaneous increased energy demand. Neither rapeseed oil nor erucic acid feeding led to electrocardiographic changes in comparison with the control sunflowerseed oil group. It is concluded that rapeseed oil and not erucic acid is responsible for loss of contractile reserve capacity without changes in the myocardial conductance system and further, that erucic acid might interfere with the peripheral vascular system. Finally, it appears that a fat rich diet might result in reduced myocardial function during a state of energy demand coupled with a blood pressure decrease.     

Deuterium isotope effect on the metabolism and toxicity of 1,2-dibromoethane

The metabolism, hepatotoxicity, and hepatic DNA damage of 1,2-dibromoethane (EDB) and tetradeutero-1,2-dibromoethane (d₄EDB) were compared in male Swiss-Webster mice. In vitro studies that measured bromide ion released from the substrate to monitor the rate of metabolism showed that the hepatic microsomal metabolism of EDB was significantly reduced by deuterium substitution, while metabolism by the hepatic glutathione S-transferases was unaffected. Three hours after ip administration of EDB or d₄EDB (50 mg/kg), there was 42% less bromide in the plasma of d₄EDB-treated mice than in the plasm of EDB-treated mice. This difference demonstrates a significant deuterium isotope effect on the metabolism of EDB in vivo. Although the metabolism of d₄EDB was less than that of EDB 3 hr after exposure, the DNA damage caused by both analogs was not significantly different at this time point. At later time points (8, 24, and 72 hr), d₄EDB caused significantly greater DNA damage than EDB. Since the decreased metabolism of d₄EDB was apparently due to a reduced rate of microsomal oxidation, these data support the hypothesis that conjugation with GSH is responsible for the genotoxic effects of EDB.     

Dietary antioxidants and the biochemical response to oxidant inhalation: I. Influence of dietary vitamin E on the biochemical effects of nitrogen dioxide exposure in rat lung

Sprague-Dawley rats derived from a specific pathogen-free colony were raised from birth on a test diet containing either 0 or 50 IU vitamin E/kg diet for 8 weeks. Rats from each dietary group were exposed to 3 ppm (5640 μg/m³) nitrogen dioxide (NO₂) continuously for 7 days. They were then killed, and the lungs analyzed for changes in weight, DNA and protein contents, tissue oxygen utilization, sulfhydryl metabolism, and the activities of NADP-reducing enzymes. The difference in dietary vitamin E alone did not cause any significant changes in these parameters. However, after NO₂ exposure the changes in these parameters relative to their corresponding unexposed controls were greater for the deficient rats than for the supplemented rats. The biochemical changes observed may be a response of the lung to injury from NO₂ exposure. The larger changes in the lungs of deficient rats may reflect a greater sensitivity of these animals to inhaled NO₂. The vitamin E contents of lung tissue in deficient and supplemented rats reflected the dietary levels. After NO₂ exposure, the vitamin E content in the lung increased significantly in supplemented rats but decreased in the deficient rats relative to their corresponding unexposed controls. The elevation of vitamin E levels in the lungs of supplemented rats with NO₂ exposure suggests its mobilization from other body sites, whereas in deficient rats this process may not have been possible.     

Gastrointestinal absorption and metabolism of capsaicin and dihydrocapsaicin in rats

Gastrointestinal absorption of capsaicin and dihydrocapsaicin was studied in rats in vivo and in situ. Rapid absorption of capsaicin or dihydrocapsaicin from stomach and small intestine occurred in vivo. About 85% of the dose was absorbed in the gastrointestinal tract within 3 hr. In situ, within 60 min after the administration of capsaicin and dihydrocapsaicin into stomach, jejunum, and ileum, about 50, 80, and 70% of the respective dose had disappeared from the lumen. When 2,4-dinitrophenol or NaCN was added, no significant reduction in uptake of [3H]dihydrocapsaicin was observed in the jejunum. These results suggested that capsaicin and its analogs were absorbed by a nonactive process in jejunum. [3H]Dihydrocapsaicin was mainly absorbed via the portal system but not a mesenteric lymphangial one. The radioactivity in the portal blood was composed of 85% of [3H]dihydrocapsaicin and 15% of its metabolite (8-methyl nonanoic acid) bound to the albumin fraction. Dihydrocapsaicin-hydrolyzing enzyme activity was found in jejunal tissue. These results suggest that capsaicin and its analogs partly received a first-pass effect, i.e., metabolism of a compound following first absorption in the gastrointestinal tract. It is concluded that capsaicin and its analogs are readily transported to the portal vein through the gastrointestinal tract by a nonactive process and partly metabolized during absorption.     

Hypolipidemic activity and toxicity studies of a styrl-hexahydroindolinol, 34–250

34–250 evoked hypocholesterolemic activity in the rat (14, 25, 31, 52, 112 mg/kg, po), dog (10, 20, 40 mg/kg, po), and monkey (30 mg/kg, po). Serum triglycerides were lowered in the rat and dog but not in the monkey. 34–250 increased [¹⁴C]acetate incorporation into liver cholesterol, but incorporation of ¹⁴C-labeled acetate into serum cholesterol was decreased. Desmosterol or 7-dehydro-cholesterol did not accumulate in serum of the three species, suggesting that inhibition of cholesterol biosynthesis by 34–250 possibly does not occur at a late stage. Normal fecal bile acid excretion was observed in rats, suggesting that cholesterol catabolism probably was not enhanced by 34–250. Compound 34–250-induced hypocholesterolemia may result from inhibition of hepatic release of this sterol into blood. The reversible hepatic lipidosis observed in rats is also possibly related to decreased hepatic transport and/or secretion of triglycerides. 34–250 did not cause a proliferation of hepatic microbodies; the lack of an increase in this fatty acid oxidizing organelle suggests that it may also have had a role in increased hepatic lipidosis. In dogs, a high incidence of severe cataracts with an early onset was induced by 20 and 40 mg/kg, po of 34–250 despite the lack of desmosterol or 7-dehydro-cholesterol in serum. The absence of these late stage intermediates of cholesterol biosynthesis in the serum of a test species does not preclude the occurrence of ocular toxicity.     

Hepatotoxicity and pulmonary toxicity of pennyroyal oil and its constituent terpenes in the mouse

Pennyroyal oil, an aromatic mint-like oil used as a flavoring and fragrance agent and as a herbal medicine, caused acute hepatic and lung damage at doses of 400 mg/kg, ip, and higher in male Swiss-Webster mice. Cellular necrosis was localized to the centrilobular regions of the liver and bronchiolar epithelial cells of the lung. Capillary gas chromatographic analysis of samples of pennyroyal oil that were obtained from health food stores showed the presence of several monoterpene constituents. R-(+)-Pulegone was the major terpene and constituted greater than 80% of the constituent terpenes in the oils that were examined. Pulegone and two other constituent terpenes, isopulegone and menthofuran, were found to be both hepatotoxic and lung toxic. Based on results of histologic scoring of necrosis, plasma GPT elevations, and hepatic glutathione depletion, R-(+)-pulegone is the terpene primarily responsible for the tissue necrosis. Furthermore, results of toxicity tests with several congeners of R-(+)-pulegone, including the enantiomeric S-(?)-pulegone, strongly implicated the α-isopropylidene ketone group as the structural unit required for eliciting hepatotoxicity, although the configurational orientation of the methyl group can modulate the hepatotoxic response.     

Effect of repeated dietary exposure of aflatoxin B1 on brain biogenic amines and metabolites in the rat

Male Sprague-Dawley rats were treated po twice weekly for 3 weeks with a low (32.8 micrograms/kg) and high dose (327.9 micrograms/kg) of aflatoxin B1 (AFB1) in corn oil. A control group received corn oil only. At the end of the experiment the rats were killed, and the concentrations of the brain catecholamines, norepinephrine (NE) and dopamine (DA), catecholamine metabolites, 3-methoxy-4-hydroxymandelic acid (VMA), homovanillic acid (HVA), and dihydroxyphenylacetic acid (DOPAC), and the indoleamine serotonin (5-HT) and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA), were determined by high-pressure liquid chromatography in five brain regions. The major effects were found in striatal dopamine and serotonin concentrations, with decreases of 37 and 29%, respectively. A corresponding decline was observed in the dopamine metabolites, homovanillic acid (44%) and dihydroxyphenylacetic acid (30%). Concentrations of these neurotransmitters and metabolites were only marginally altered in cerebral cortex, cerebellum, hypothalamus, and medulla oblongata. It appears that a major effect of AFB1 is on dopaminergic pathways, possible by selectively perturbing the conversion of tyrosine to biogenic catecholamine neurotransmitters.     

Effect of the organic acid transport inhibitor probenecid on renal cortical uptake and proximal tubular toxicity of hexachloro-1,3-butadiene and its conjugates

Hexachloro-1,3-butadiene (HCBD), its glutathione conjugate (HCBD-GSH), cysteine conjugate (HCBD-CYS), and mercapturic acid derivative (HCBD-NAC) all produce acute necrosis of the pars recta of the proximal renal tubule in the rat. Previous studies have shown that radiolabel from administered HCBD appears to concentrate in the pars recta region. Renal uptake of radioactivity from HCBD-NAC was studied in rats by giving a single ip injection of the chemical and measuring its concentration in plasma and renal cortex 4 hr later. Cortex/plasma ratios (C/P) of HCBD-NAC were 4.35 +/- 0.21 (8 animals) at a dose of 64 mumol/kg and 10.4 +/- 0.55 (5) at a dose of 16 mumol/kg. These ratios were greater than that of inulin [C/P inulin = 1.5 +/- 0.2 (4)]. Thus cortical HCBD-NAC content was significantly greater than can be accounted for by glomerular filtration alone. Prior administration of probenecid (500 mumol/kg), a competitive inhibitor of organic acid transport, to animals receiving 16 or 64 mumol/kg of HCBD-NAC reduced the C/P to 1.03 +/- 0.09 (5) and 0.81 +/- 0.05 (8), respectively. Administration of probenecid in increasing doses (100, 200, 300, and 400 mumol/kg) to animals receiving 64 mumol/kg HCBD-NAC resulted in decreases of the C/P (2.59, 2.29, 1.35, and 0.84, respectively), suggesting a competitive inhibition of cortical HCBD-NAC uptake. The extent of covalently bound radioactivity from 64 mumol/kg HCBD-NAC was significantly greater in the renal cortex (1.11 +/- 0.2 nmol eq/mg protein) than in the liver (0.19 +/- 0.01 nmol eq/mg protein). Prior administration of probenecid (500 mumol/kg) reduced the renal cortical concentration of HCBD-NAC to 0.25 +/- 0.02 nmol eq/mg protein. Increasing doses of probenecid resulted in a progressive decrease in renal cortical covalent binding. When treatment with probenecid led to renal cortical concentrations of less than 120 nmol eq HCBD-NAC/g and an amount of covalently bound material less than 0.4 nmol eq/mg protein the animals were completely protected against the nephrotoxicity, as assessed by plasma urea and histopathological examination 24 hr after dosing. Prior administration of probenecid (500 mumol/kg) also protected rats against the nephrotoxicity produced by HCBD (192 mumol/kg), HCBD-GSH (47 mumol/kg), and HCBD-CYS (36 mumol/kg). It is suggested that the renal cortical accumulation and selective proximal tubular toxicity of HCBD and its conjugates is related to a carrier-mediated transport system.     

The chronic toxicity of technical and analytical pentachlorophenol in cattle. I. Clinicopathology

The objectives of this study in female yearling Holstein cattle were to define the toxic effects of (1) long-term exposure to analytical pentachlorophenol (aPCP), and (2) to determine the influence of the contaminants in technical pentachlorophenol (tPCP) in the toxic syndrome. Four groups of three heifers each were exposed for 160 days to aPCP, tPCP, or a mixture thereof in the feed. A fifth group of three animals served as unexposed controls. All treated cattle received the same amount of PCP; 20 mg/kg/day for 42 days which was reduced to 15 mg/kg/day for the remainder of the study because of a suspected decrease in body weight gain in all PCP-exposed animals compared to controls. Fat and liver samples for chemical analyses were collected at the end of the study. Major findings in the tPCP-exposed heifers included a dose-related decrease in body weight, decreased feed efficiency, progressive anemia, a dose-related increase in liver and lung weights, and a decrease in thymus weight. The most conspicuous lesion was market villous hyperplasia of the urinary bladder mucosa in two of three animals exposed to the highest level of tPCP. There were minimal hepatic lesions although hyperplasia of the mucosal lining of the gali bladder and bile duct was noted in some animals exposed to tPCP. Animals exposed to aPCP were, in general, comparable to the controls. Hepatic mixed function oxidases were increased by aPCP, but more so by tPCP. A decrease in thyroxine concentration was found in all PCP-treated cattle. Immunologic studies suggested a progressive tPCP dose-related enhancement in the lymphoproliferative response, an in vitro correlate for cell-mediated immunity. Observed effects on humoral immune parameters were equivocal. The results of this study indicate that toxicity of PCP in cattle is primarily attributable to its contamination with toxic impurities.     

Effect of various metabolic inhibitors on biphenyl metabolism in isolated rat hepatocytes.

The effect of three inhibitors of mitochondrial function (menadione, rotenone and 2,4-dinitrophenol) on drug metabolism in isolated rat hepatocytes has been studied. Menadione (at 1.25 × 10^?4 M) caused almost complete inhibition of biphenyl Phase I metabolism whereas rotenone (2 × 10^?5 M) inhibited the same reaction only by 25 per cent although the subsequent conjugation of the Phase I metabolite was markedly depressed. Qualitatively similar findings were observed with hepatocytes isolated from phenobarbital-pretreated rats, and with liver microsomes isolated from control rats. 2,4-Dinitrophenol (2 × 10^?4 M) caused a marked enhancement of biphenyl Phase I metabolism but a marked inhibition of subsequent conjugation. This enhancement of Phase I metabolism was not observed in control cells with other substrates (benzo[a]pyrene, 7-ethoxycoumarin), nor in biphenyl metabolism in “induced” cells or in liver microsomes isolated from control rats. It is tentatively suggested that products of 2,4-dinitrophenol metabolism may “activate” biphenyl metabolism in intact liver cells. Furthermore, it is suggested for all three inhibitors that direct effects on the drug metabolizing enzyme systems (Phase I and Phase II) are as important as their effects on mitochondrial function in explaining their inhibition of drug metabolism. It appears that Phase II metabolism of xenobiotics is more susceptible to inhibition by metabolic inhibitors than is Phase I metabolism, probably due to depletion of the cellular ATP levels.     

Differential toxicity of trans-permethrin in rainbow trout and mice: I. Role of biotransformation

The trans isomer of [1R,S] permethrin (t-per) was > 110 times more toxic to rainbow trout than to mice by both iv and ip administration. The importance of trans-permethrin biotransformation in this differential toxicity was assessed by measuring rates of t-per biotransformation in trout and mouse tissues in vitro, and the effect of inhibitors of drug metabolism on t-per lethality in both species. A previous study had shown that ester hydrolysis by trout liver, plasma, and kidney is much slower than that seen in these same tissues in mice. The present work further indicates that oxidation of t-per is 35 times slower in trout liver microsomes than mouse microsomes when the tissue suspensions were incubated at the body temperature of trout and mice (12°C for trout and 37°C for mice). Inhibition of esterase activity with tri-o-tolyl phosphate (TOTP) produced no potentiation of t-per lethality in trout while the same compound potentiated t-per lethality at least 1.5-fold in mice. Piperonyl butoxide (PIP) alone produced no potentiation in mice but slightly increased t-per toxicity when administered in conjunction with TOTP. PIP caused a slight increase in t-per lethality in rainbow trout but no increase in t-per lethality from control was observed when trout were pretreated with both TOTP and PIP. When drug metabolism was inhibited, t-per was still 65 times more toxic to trout than to mice. The data indicate that trout, in addition to hydrolyzing t-per slowly, also oxidize the compound considerably slower than mice in vitro. Potentiation of t-per lethality by TOTP suggests ester hydrolysis to be an important t-per detoxification reaction in mice but not in trout. However, since t-per was 65 times more toxic to the trout than mouse when drug metabolism was inhibited, other factors, such as differences in target organ sensitivity may be involved in the differential toxicity of permethrin.     

Differential toxicity of trans-permethrin in rainbow trout and mice: II. Role of target organ sensitivity

Previous studies demonstrated that even when trans-permethrin ester hydrolysis was inhibited in mice and the toxicity of trans-permethrin was increased at least 1.6 times by tri-o-tolyl phosphate (TOTP), the toxicity of trans-permethrin remained 60 times greater in the rainbow trout than in the mouse. This information suggested that factors other than metabolism, such as target organ sensitivity, may play a role in the differential toxicity of trans-permethrin between rainbow trout and mice. Since the brain of both the fish and the mouse is believed to be a site of action of the pyrethroids, the concentration of permethrin in the brains of these two species at the onset of signs of pyrethroid toxicity was measured in an attempt to establish a correlation between permethrin brain levels and toxicity in the fish and mammal. Signs of pyrethroid toxicity, such as intense whole body twitches and loss of equilibrium, were observed in rainbow trout when ¹⁴C-trans-permethrin brain levels exceeded 1.5 μg/g brain, regardless of whether the compound was administered iv or ip. Pyrethroid toxicity signs, such as intense tremors and loss of righting, were not observed in mice until ¹⁴C-trans-permethrin levels reached 25 to 30 μg/g brain. Thin-layer chromatographic analysis indicated that 85 to 95% of the ¹⁴C was parent compound in the rainbow trout brains compared to 60 to 70% in the mouse brains. ¹⁴C-cis-Permethrin produced lethality in trout and mice when the ¹⁴C-cis-permethrin brain concentrations were 2 and 6 μg/g, respectively. The results suggest that the rainbow trout may be physiologically more sensitive to the action of permethrin than is the mouse. In the trout, the cis and trans isomers appear to be equal in toxicity while in the mouse the cis isomer is more active than the trans isomer. The difference in isomer sensitivity between the trout and mouse may suggest differences in specificities at the site of pyrethroid action. The physiologic basis for this difference in pyrethroid toxicity is not understood.