Protein synthesis by hepatic mitochondria isolated from carbon tetrachloride-exposed rats.

Hepatic mitochondria isolated from rats 40 h after dosage with 1.1 ml/kg CCl4 are uncoupled and display structural damage. Mitochondrial function returns during hepatic recovery. Because the products of mitochondrial protein synthesis are essential to mitochondrial structure and function, the effects of CCl4 on the rate of mitochondrial protein synthesis, and on the products, was studied using mitochondria from CCl4-exposed rats during the early, maximum development and resolution stages of CCl4-induced mitochondrial damage. Rates of mitochondrial protein synthesis (incorporation of [35S]methionine) were elevated 300% over that of mitochondria from non-exposed rats 17 h after exposure; depressed by 50% at 40 h and above control at 113 h. When the radiolabeled products of incorporation were separated and examined by autoradiography, a novel, low-molecular-weight band, of approx. 9700, was apparent 40 h after CCl4 exposure. A band of similar molecular weight appeared when control mitochondria were incubated without an exogenous supply of ATP. Mitochondria from exposed rats which displayed rates of protein synthesis greater than control consistently had a relative increase in a band that corresponded in size to that of cytochrome oxidase subunit I. It was concluded that the loss of mitochondrial function induced by CCl4 could not be attributed to inhibition of mitochondrial protein synthesis, and that the mitochondria may not always synthesize protein in constant proportions.

Hyperbaric pressure of 51 atmospheres is without effect on the depression of oxygen uptake in kidney tissue culture produced by halothane.

Although anesthetized animals are awakened when subjected to increased pressure, compression does not result in antagonism of all phenomena associated with these drugs. It has recently been demonstrated that halothane's inhibition of respiration of isolated rat liver mitochondria is not reversed by hydraulic compression to 51 atmospheres. In order to determine whether this phenomenon can be extrapolated to the whole cell, we have investigated the effect of hydraulic compression of intact renal cells equilibrated with halothane, and conclude that pressure does not overcome the inhibitory effect of this anesthetic.

Carbon tetrachloride depresses hepatic phospholipid synthesis in rats.

40 h after an acute dose of CCl4 (11.3 mmol/kg), the incorporation of [1-3H]ethanolamine into rat hepatic microsomal phospholipids was inhibited to 70% of control. Incorporation into phospholipids of the inner and outer mitochondrial membranes was 30-35% of control. Rates of incorporation were equal to or above normal rates in all membranes 65 h after dosage. The activity of methyltransferase in microsomal fractions isolated from rats 10 to 66 h after dosage was depressed. These data suggest that the alteration of mitochondrial phospholipids that parallels mitochondrial dysfunction after acute CCl4 dosage could be attributed to a CCl4-induced inhibition of the microsomal phospholipid biosynthetic pathways.

Binding of carbon tetrachloride metabolites to rat hepatic mitochondrial DNA.

Radioactivity from [14C]CCl4 was bound to highly purified mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) prepared from livers of rats after a single dose of [14C]CCl4. At a low, non-necrotizing dose as well as at an acutely toxic dose, mtDNA bound 20-50-fold more radioactivity per mg than did nDNA. Extensive enzymatic digestion and purification of mtDNA did not remove radioactivity. Binding of radioactivity to mtDNA could also be demonstrated after anaerobic incubation of isolated mitochondria with [14C]CCl4, NADPH, ADP, and succinate. Our results suggest that CCl4 can be activated by rat hepatic mitochondrial enzymes to metabolites which bind covalently to mtDNA.

Methoxyacetic acid and ethoxyacetic acid inhibit mitochondrial function in vitro.

Ethylene glycol monomethyl ether (EGME) and ethylene glycol monoethyl ether (EGEE) have recently been shown to be potent reproductive toxicants in laboratory animals. The toxicity of these compounds is believed to be due to their metabolites, methoxyacetic acid (MAA) and ethoxyacetic acid (EAA). Since the primary targets of EGME and EGEE appear to be tissues with rapidly dividing cell systems and high rates of respiration and energy metabolism, the effects of these compounds and their proposed metabolites on mitochondria were investigated. At concentrations beginning at 3.85 mM, MAA and EAA inhibited state 3 respiration and the respiratory control ratio (RCR) in hepatic mitochondria with either succinate or citrate/malate as substrates. Cytochrome c oxidase activity was also inhibited by both metabolites at similar concentrations. The effects of MAA, the metabolite from the more potent compound, on testicular mitochondria were found to be comparable. Neither EGME or EGEE appeared to affect mitochondrial function at concentrations as high as 238 or 113 mM, respectively. These results support the hypothesis that the toxicity of EGME and EGEE are due to their metabolites, MAA and EAA, and that these metabolites may exert their effects, in part, on mitochondrial function.

Effect of lead acetate on Sertoli cell lactate production and protein synthesis in vitro.

The effects of lead acetate on protein synthesis and lactate production by cultures of rat Sertoli cells in vitro were studied. Sertoli cell cultures prepared from 20 day old Sprague-Dawley rats were exposed to 0.01, 0.05 and 0.10 mM lead acetate. Lactate production was significantly elevated by all concentrations of lead after 3, 6, 9 and 12 hours of exposure. Protein biosynthesis as measured by [3H]-leucine incorporation was significantly depressed by 0.05 and 0.10 mM lead acetate after 2 hours of exposure. These results support the hypothesis that lead acetate may inhibit spermatogenesis by a disturbance of the metabolic activities of the Sertoli cells.

Mutagen activation of 1,2-dibromo-3-chloropropane by cytosolic glutathione S-transferases and microsomal enzymes.

It is not clear whether glutathione (GSH) conjugation to 1,2-dibromo-3-chloropropane (DBCP) results in genotoxic activation. Therefore S9, cytosolic, and microsomal fractions from uninduced rat liver were evaluated for their relative ability to activate DBCP in a modified Ames system. The S9 enzymes, either alone or in combination with exogenous GSH, did not enhance the mutagenicity of DBCP; identical results were obtained with cytosolic enzymes. Significant mutagenic activation of DBCP was produced by either S9 or microsomal fractions in the presence of NADPH. Activation was proportional to cytochrome P-450 concentrations, and was diminished by exogenous GSH. The protection against genotoxicity exerted by GSH did not require cytosolic glutathione S-transferases (GST). Thus, mutagenic activation of DBCP as obtained with S9 fractions is primarily due to biotransformation by microsomal rather than by cytosolic enzymes. Kinetic studies of cytosol-catalyzed conjugation of GSH to DBCP revealed tissue-specific differences in apparent Km and Vmax. Renal and testicular GSTs were associated with 28-46% smaller Vmax values when compared to hepatic GSTs (31.2 +/- 1.9 nmol/min X mg protein). However, renal and testicular GSTs had relatively higher affinities for DBCP. Thus, extrahepatic tissues possess significant capacity to conjugate GSH to DBCP. DBCP-GSH conjugates may undergo enzymatic modification by extrahepatic peptidase and beta-lyase to yield other sulfur-containing moieties that perhaps mediate DBCP's extrahepatic toxicity.

The effect of 2-methoxyethanol and methoxyacetic acid on Sertoli cell lactate production and protein synthesis in vitro.

Exposure to 2-methoxyethanol (ME) or its major metabolite, methoxyacetic acid (MA), results in spermatocyte depletion and testicular atrophy in experimental animals. The site of spermatogenesis is within the seminiferous tubule. Sertoli cells support spermatogenesis, synthesizing and secreting proteins, and metabolic substrates for utilization by differentiating germ cells in the seminiferous tubule lumen. One of these substrates, lactate, is preferentially metabolized by spermatocytes. Therefore, because germ cells are dependent upon the metabolic products of Sertoli cells, the effect of ME and MA on production of lactate and protein synthesis was measured in cultured rat Sertoli cells. Cell cultures were incubated with ME or MA at 0, 3, or 10 mM for up to 12 hr. No significant difference was seen in total protein synthesis as measured by [3H]leucine incorporation. ME and MA had no apparent effect on cell viability. However, lactate concentrations and rates of lactate accumulation were significantly decreased by MA, but not ME, at both 3 and 10 mM following incubation for 6, 9, and 12 hr. The results suggest that inhibition of Sertoli cell lactate production resulting from ME or MA exposure could account for the inhibitory action of these compounds on spermatogenesis.

Effects of methanol vapors on testosterone production and testis morphology in rats.

Potential toxic effects of methanol vapors on testicular production of testosterone and the morphology of testes were investigated using normal or methanol-sensitive folate-reduced rats. Methanol inhalation at the level of the current permissible exposure limit, 200 ppm, for up to six weeks (8 hours/day, 5 days/week), did not reduce serum testosterone levels in normal rats. Testes isolated from methanol-exposed (200 ppm) rats had the same capability as those from air-exposed rats in synthesizing testosterone whether testes were incubated in the absence or presence of hCG. The testes-to-body weight ratio of rats exposed up to 800 ppm methanol for up to 13 weeks (20 hours/day, 7 days/week) were not different from those of the air-exposed rats. Furthermore, methanol had no adverse effect on testicular morphology at the end of the 13 week exposure period at 800 ppm in either normal rats or folate-reduced methanol-sensitive rats when they were 10 months old at the time of examination. Thus, these data indicate that low level methanol may not cause an inhibitory effect on testosterone synthesis contrary to previous literature reports. However, a greater incidence of testicular degeneration was noticed in the 18 month old folate-reduced rats exposed to 800 ppm for 13 weeks (20 hours/day, 7 days/week), suggesting that methanol may have a potential to accelerate the age-related degeneration of the testes.

Modification of methanol potentiation of CCl4 toxicity in rats by chloramphenicol and salicylate.

The mechanisms by which methanol potentiates CCl4 hepatotoxicity was studied in rats. Chloramphenicol, an inhibitor of cytochrome P-450, blocked the increase of serum glutamate-oxaloacetate transaminase activity enhanced by methanol pretreatment of rats exposed to CCl4. Chloramphenicol also decreased microsomal lipid peroxidation in both CCl4 and methanol-pretreated, CCl4-intoxicated animals when measured 30 minutes after exposure. Chloramphenicol prevented the loss of glucose 6-phosphatase activity after CCl4 and methanol. Sodium salicylate, which lowers the level of NADPH in the hepatocyte, blocked methanol potentiation of CCl4 damage as measured by the elevation of serum GOT activity. Therefore, methanol may potentiate CCl4 hepatotoxicity by stimulation of CCl4 bioactivation by cytochrome P-450 via an increase in the level of reduced NAD(P)H in the liver.

Effect of general anesthetics and pressure on aerobic metabolism of monkey kidney cells.

The authors examined the inhibition of aerobic metabolism in monkey kidney cell cultures exposed to halothane, enflurane, and isoflurane. The ability of hyperbaric pressure to reverse the halothane-induced metabolic inhibition also was examined. Incubation of two monkey kidney cell lines for 24 h with clinically equipotent concentrations (2.6 MAC) of halothane, enflurane, or isoflurane vapors increased the concentration of lactate in the media by 126 to 244% relative to nonexposed control cultures. The increased rate of lactate accumulation was proportional to the concentration of halothane and was accompanied by a decrease in media pH. Removal of halothane restored the normal rate of lactate production. Hyperbaric pressures of 25, 50, and 100 atmospheres did not alter the halothane-stimulated rate of lactate production relative to non-anesthetic-treated controls, although pressure alone did depress the rate of lactate accumulation in all cultures. The stimulation of lactate production likely reflects the known ability of halothane to inhibit mitochondrial respiration. The failure of pressure to reverse the stimulation of lactate production by halothane suggests that inhibition of mitochondrial metabolism cannot be reversed by pressure.

Primary rat Sertoli and interstitial cells exhibit a differential response to cadmium.

Two cell types central to the support of spermatogenesis, the Sertoli cell and the interstitial (Leydig) cell, were isolated from the same cohort of young male rats and challenged with cadmium chloride to compare their susceptibility to the metal. Both cell types were cultured under similar conditions, and similar biochemical endpoints were chosen to minimize experimental variability. These endpoints include the uptake of 109Cd, reduction of the vital tetrazolium dye MTT, incorporation of 3H-leucine, change in heat-stable cadmium binding capacity, and production of lactate. Using these parameters, it was observed that the Sertoli cell cultures were adversely affected in a dose-and time-dependent manner, while the interstitial cell cultures, treated with identical concentrations of CdCl2, were less affected. The 72-hr LC50's for Sertoli cells and interstitial cells were 4.1 and 19.6 microM CdCl2, respectively. Thus, different cell populations within the same tissue may differ markedly in susceptibility to a toxicant. These in vitro data suggest that the Sertoli cell, in relation to the interstitium, is particularly sensitive to cadmium. Because the Sertoli cell provides functional support for the seminiferous epithelium, the differential sensitivity of this cell type may, in part, explain cadmium-induced testicular dysfunction, particularly at doses that leave the vascular epithelium intact.

Comparative theobromine metabolism in five mammalian species.

Biotransformation of theobromine (TBR) was compared in rats, mice, hamsters, rabbits, and dogs by assaying urinary metabolites using HPLC after oral administration of a 5 mg/kg dose containing 8-14C-TBR. Recovery of radioactivity ranged from 60-89% of the dose in urine, and from 2-38% of the dose in feces, with most material being excreted during the first 48 hr after dosing. TBR was most extensively metabolized by rabbits and male mice. The primary metabolite excreted by rats and mice was 6-amino-5-[N-methylformylamino]-1-methyluracil (6-AMMU); male mice converted TBR to this metabolite more extensively than did female mice. Rabbits and dogs metabolized TBR primarily to 7-methylxanthine (7-MX) and 3-methylxanthine (3-MX), respectively; the major metabolites excreted by hamsters were 6-AMMU and 7-MX. Overall N-demethylase activity yielding monomethyl metabolites was greatest in rabbits and lowest in rats. Ring N-demethylation at position 3 predominated over 7-N-demethylation in all species except the rat and dog. In dogs, TBR was N-demethylated primarily at position 7, while N-demethylase activity in rats was without apparent positional specificity. Oxidation of methylated xanthines to the corresponding uric acids was a relatively minor metabolic pathway in all species, but had greatest activity in mice. Oxidation of TBR to 3,7-dimethyluric acid was significantly greater in female rats than in male rats. In summary, excretion patterns of TBR and its metabolites were qualitatively similar among species, indicating that TBR is metabolized along similar pathways. Except for the excretion of small quantities of an unidentified but apparently unique metabolite by dogs, only quantitative species- and sex-related differences were observed in the metabolic disposition of TBR.