Metabolism and excretion of piperonyl butoxide in the rat.

[14C]-piperonyl butoxide (PBO) was administered to male and female rats by gavage at a dose rate of 50 or 500 mg kg-1 body weight. In all cases, the radioactivity was rapidly excreted with 87-99% being found in the 0-48-h excreta and the majority of the dose (64.1-85.0%) being eliminated in faeces. The metabolism of PBO was complex with over 25 peaks of radioactivity being seen by radio-high-performance liquid chromatography (HPLC). Using HPLC/tandem mass spectrometry (MS/MS) and nuclear magnetic resonance (NMR), 12 urine metabolites were assigned structures together with four plus PBO in faeces. Metabolism occurred at two sites: the methylenedioxy ring, which opened to form a catechol that could then undergo methylation, and the 2-(2-butoxyethoxy)ethoxymethyl side-chain, which underwent sequential oxidation to a series of alcohols and acids. The identified metabolites accounted for approximately 60% of the administered dose.

The impact of rice pesticides on the aquatic ecosystems of the Sacramento River and Delta (California).

Since the early 1980s, when molinate was demonstrated to have killed carp in agricultural drains, an intensive research effort has been undertaken to assess the impact of rice pesticides on aquatic ecosystems in the Sacramento River and Delta. No impact has been found that can be clearly attributed to rice pesticides. However, the rice insecticides methyl parathion and carbofuran, and probably also bufencarb, reached levels in the River and Delta that, based on laboratory bioassays, would have been toxic to aquatic microinvertebrates and, in the case of bufencarb, to early life stages of striped bass. Reductions in microinvertebrate populations could have impacted higher organisms in the aquatic food chain such as striped bass and chinook salmon. Bufencarb was not used after 1981. Since then, changes in the management of the remaining rice pesticides have resulted in dramatic decreases in the levels of these chemicals in the River and Delta. Levels achieved today have no known toxicity to aquatic organisms. As releases of rice pesticides were reduced to achieve nontoxic levels in the River and Delta, however, commensurate recoveries of striped bass and chinook salmon did not occur, suggesting that rice pesticides may have had little or no role in the decline of these species.

The toxicological significance of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds in human adipose tissue.

Reports of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzo-furans (PCDFs) in human tissues were reviewed to assess their toxicological significance. The predominance of 2,3,7,8-chlorinated congeners in human tissues and in the food chain, but not in other environmental matrices, suggests that the food chain is the major source of human residues. Exposures to unique distributions of congeners can result in recognizable patterns of excess 2,3,7,8-chlorinated PCDDs/PCDFs in humans. Current levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) in the general population can be accounted for by an average level of 133 or 27 ppq (parts per quadrillion) in food based on an estimated half-life in humans of 1 or 5 yr, respectively. 2,3,7,8-TCDD is more persistent in humans than in rodents or lagomorphs, resulting in higher body burdens in humans at comparable levels in the diet. Taken alone, this toxicokinetic difference would increase risks estimated for humans from toxicity in laboratory animals. However, humans appear to be less susceptible due to the following: less food is ingested per body mass, more 2,3,7,8-TCDD is sequestered in adipose tissue and away from target organs, and tissue susceptibility appears to be lower than in the most sensitive rodents and lagomorphs. The body burden of 2,3,7,8-TCDD in a Seveso woman receiving an apparently nontoxic dose was approximately 180 times the average body burden of 2,3,7,8-TCDD equivalents in the general population of industralized societies. The body burden of prisoners who were exposed dermally to a suspension of 2,3,7,8-TCDD and who developed severe chloracne was estimated to be as much as 38 times that of the Seveso woman. These comparisons suggest a considerable margin of safety for the general population.

Comparative metabolism and toxicity of chemical carcinogens in primary cultures of hepatocytes.

Hepatocytes were prepared by an in situ or biopsy perfusion of liver with collagenase. Hepatocytes from adult liver were cultured without serum on collagen-coated dishes in culture medium supplemented with hormones. Stable monolayers were established within 24 h and were maintained for up to 10 d. The hormone supplement maintained cytochrome P-450, a critical component of mixed function oxygenase responsible for activation of many procarcinogens. The addition of serum and phenobarbital to the cultures also maintained higher levels of mixed function oxygenase activity. Viable cultures were prepared from mice, rats, guinea pigs, rabbits, dogs, monkeys, and humans. Metabolism studies revealed the rate of metabolism and the extent of covalent binding to macromolecules, including DNA. Measures of cytotoxicity and genotoxicity in vitro provide an indication of hepatonecrotic and hepatocarcinogenic potency in vivo. Comparative metabolism, cytotoxicity, and genotoxicity studies provide a means of facilitating the extrapolation of toxicity data from laboratory animals to humans.

Enhancement of hepatocellular genotoxicity of several mutagens from amino acid pyrolysates and broiled foods following ethanol pretreatment.

The effect of subchronic ethanol ingestion on the genotoxicity and metabolism of the mutagens 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,5-b]indole (Trp-P-2), 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), 2-aminodipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4- dimethylimidazo[4,5-f]quinoline (MeIQ) was evaluated in primary cultures of rat hepatocytes. Male Sprague-Dawley rats were pair-fed, for 8 days, liquid diets containing either ethanol (8%, v/v) or an isocaloric sucrose solution. Ethanol pretreatment significantly (P less than 0.05, Student's t test) enhanced the level of DNA repair stimulated by Glu-P-1, Glu-P-2, IQ and MeIQ. Statistically significant increases in DNA-repair activity ranged from 1.9-fold for IQ to 3.4-fold for Glu-P-2. Following a 16-hr exposure, the concentration of parent mutagen in the culture medium decreased by 75-98%. Neither the rate of mutagen metabolism in hepatocyte cultures nor the extent of mutagenic activation in microsome preparations was appreciably affected by ethanol pretreatment. The results suggest that ethanol pretreatment enhances the genotoxicity of Glu-P-1, Glu-P-2, IQ and MeIQ by inducing non-microsomal activation processes.

Acetaminophen metabolism, cytotoxicity, and genotoxicity in rat primary hepatocyte cultures.

Acetaminophen (APAP) metabolism, cytotoxicity, and genotoxicity were measured in primary cultures of rat hepatocytes. Although 3 mM APAP caused a slight increase in cellular release of lactate dehydrogenase into the culture medium, cellular glutathione concentration (an index of APAP metabolism) was reduced by 50%. APAP at 7 mM was significantly more toxic to these hepatocytes and had a similar but more marked effect on glutathione concentrations. In spite of its cytotoxicity, neither dose of APAP stimulated DNA repair synthesis when monitored by the rate of incorporation of [3H]thymidine into DNA following exposure to APAP. Thus, although APAP has been shown to be both hepato- and nephrotoxic in several in vivo and in vitro systems, the reactive toxic metabolite of APAP is not genotoxic in rat primary hepatocyte cultures.

Genotoxicity of the cooked-food mutagens IQ and MeIQ in primary cultures of rat, hamster, and guinea pig hepatocytes.

To investigate possible interspecies differences in the hepatocellular genotoxicity of the food-borne mutagens 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethyl-imidazo[4,5-f]quinoline (MeIQ), primary cultures of rat, hamster, and guinea pig hepatocytes were established. The induction of DNA repair activity in cultures exposed to various concentrations of IQ and MeIQ was determined by liquid scintillation spectrometry. DNA repair responses to MeIQ were, in general, greater than those elicited by IQ. In all three preparations of rat and hamster hepatocytes and in two of three preparations of guinea pig cells, MeIQ produced statistically significant (p less than 0.05) repair responses. IQ stimulated significant levels of repair in all three rat hepatocyte preparations and in two of three hamster cell preparations. In guinea pig cells exposed to IQ, no significant repair activity was observed. These results indicate that the genotoxicity of IQ and MeIQ in hepatic cells in species-dependent.

Effects of butylated hydroxytoluene pretreatment on the metabolism and genotoxicity of aflatoxin B1 in primary cultures of adult rat hepatocytes: selective reduction of nucleic acid binding.

To elucidate biochemical mechanisms underlying the anticarcinogenic activity of butylated hydroxytoluene (BHT), studies were undertaken to characterize the influence of BHT pretreatment on the metabolism and genotoxicity of aflatoxin B1 (AFB1) in primary cultures of rat hepatocytes. During a 10-day pretreatment period, adult male rats were fed either a control diet or a diet supplemented with 0.5% BHT. Hepatocytes were subsequently isolated from each animal and cultured in chemically defined medium. Cultures prepared from rats which had been fed BHT metabolized AFB1 more rapidly than did controls. BHT pretreatment also enhanced oxidation of AFB1 to aflatoxin M1 (AFM1), and accelerated the rate of AFM1 conjugation. Covalent binding to DNA and RNA in BHT-pretreated cultures was reduced by 91 and 82%, respectively, while protein binding decreased by only 29%. AFB1 did not stimulate detectable DNA repair synthesis in BHT-pretreated cultures, although stimulation of DNA repair was clearly evident in control cultures. In a separate experiment, consistently higher baseline concentrations of reduced glutathione were observed in BHT-pretreated cells, indicating that BHT pretreatment may enhance formation of detoxified glutathione conjugates of AFB1. These findings suggest that the anticarcinogenic activity of BHT is due in part to preferential enhancement of hepatic detoxification mechanisms, with the result that intracellular concentrations of reactive metabolites are reduced and fewer covalently bound adducts are formed.

Genotoxicity of N-nitrosothiazolidine in microbial and hepatocellular test systems.

The genotoxicity of N-nitrosothiazolidine was studied in a microbial system and in a hepatocellular system. The compound showed positive responses in both tests, exhibiting weak mutagenicity at the lowest level tested (1 mg/plate) in the rec assay in Bacillus subtilis, and inducing statistically significant levels of DNA repair in primary hepatocyte cultures at concentrations ranging from 2 X 10(-4) to 2 X 10(-3) M.

Effect of O2 tension on the bioactivation and metabolism of aliphatic halides by primary rat-hepatocyte cultures.

The covalent binding of CCl4 and CF3CHBrCl to cultured rat hepatocytes was enhanced by anoxic conditions, indicative of enhanced reductive biotransformation. The covalent binding of CHCl3 and CHCl2CH2Cl decreased as the O2 concentration was decreased, indicating a preference for oxidative metabolism. Lower O2 tensions decreased the formation of polar water-soluble metabolites with CHCl3 and CHCl2CH2Cl, while CCl4 and CF3CHBrCl were unaffected. The results indicate that primary cultured hepatocytes can reductively biotransform and bioactivate aliphatic halides under anoxic conditions.

The effect of phenobarbital pretreatment on the metabolism, covalent binding, and cytotoxicity of aflatoxin B1 in primary cultures of rat hepatocytes.

The effect of phenobarbital (PB) pretreatment on the metabolism, covalent binding, and cytotoxicity of [14C]aflatoxin B1 (AFB1) was studied in primary hepatocyte cultures. Hepatocytes from control and PB-pretreated rats were isolated from perfused liver biopsies and cultured in a chemically defined, hormone-supplemented medium. [14C]AFB1, dissolved in medium, was added to cultures at 20-22 h. The metabolism of AFB1 to water-soluble products and its binding to trichloroacetic acid-precipitable macromolecules were assessed 0.5 to 24 h later. At 6 h, PB pretreatment reduced total binding to macromolecules by 31% and reduced binding to RNA and DNA by 61% and 66%, respectively. In addition, PB protected cultures from the cytotoxic effects of AFB1, as evidenced by a significantly reduced (p less than 0.05) leakage of lactate dehydrogenase into the medium at 51 h. Elevated mixed-function oxidase and glutathione S-transferase activities, as well as higher levels of AFB1-glutathione conjugate were measured in cultures from rats pretreated with PB. The protective action of PB was concluded to be due to the induction of hepatic glutathione S-transferases responsible for the detoxification of AFB1.

Metabolism of food toxicants: saccharin and aflatoxin B1, a contrast in metabolism and toxicity.

The artificial sweetener, saccharin, and the secondary mold product, aflatoxin B1, are present in many foods consumed by humans. Both chemicals produce cancer in rats. For this reason, they have aroused concern among scientists and the public as to the carcinogenic risk that they pose to humans. A comparison of these two chemicals reveals striking contrasts in potency, metabolism, mechanism-of-action, and experimental approach to assessing metabolism. These contrasts are examined in detail to illustrate the importance of metabolism in safety evaluation.

Aroclor 1254 pretreatment enhances the DNA repair response to amino acid pyrolysate mutagens in primary cultures of rat hepatocytes.

The stimulation of unscheduled DNA synthesis (UDS) by 3-amino-1,4-dimethyl -5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) and 2-amino-dipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2) was measured in primary cultures of rat hepatocytes. Hepatocellular DNA was recovered as a cetyltrimethylammonium salt from nuclei digested with protease. The incorporation of [3H]thymidine was measured by liquid scintillation counting techniques. Although Trp-P-1 was the most potent of the pyrolysis products tested, it was considerably less active than 2-aminofluorene (2-AF) or aflatoxin B1 (AFB1). A greater induction of UDS by Trp-P-1 was observed in cultures from female rats than in cultures from male rats. Aroclor 1254 pretreatment resulted in a substantially greater UDS response to all 4 pyrolysis products.

The comparative metabolism and toxic potency of aflatoxin B1 and aflatoxin M1 in primary cultures of adult-rat hepatocytes.

Both aflatoxin B1 (AFB1) and a hydroxylated metabolite, aflatoxin M1 (AFM1), were potent cytotoxins and genotoxins to primary cultures of rat hepatocytes. However, AFB1 stimulated the release of lactate dehydrogenase into the culture medium and the loss of viable cells from the monolayer at lower doses than did AFM1. The lowest toxic doses of AFB1 and AFM1 were 0.05-01 and 0.6 microgram/culture, respectively. Genotoxicity, determined by an assay for stimulation of DNA repair, was apparent at lower doses than was cytotoxicity. AFB1 was again more potent than AFM1, stimulating DNA repair at 0.025 microgram/culture, compared to the lowest genotoxic dose of AFM1 of 0.05 microgram/culture. At higher doses (1.2-2.4 microgram/culture) the responses due to both aflatoxins in the cytotoxicity and DNA-repair assays were approximately equal. The metabolism of a low dose (c. 0.17 microgram/culture) of [14C]AFB1 and [3H]AFM1 by cultured hepatocytes differed significantly. After 1 hr, 50% of the [14C]AFB1 remained unchanged in the culture medium, whereas about 18 hr were required for the same amount of [3H]AFM1 metabolism to occur [14C]AFB1 was metabolized to AFM1, to polar metabolites recovered in the aqueous phase after chloroform extraction, and to metabolites covalently bound to hepatocyte macromolecules. [3H]AFM1 was also metabolized to polar metabolites and to forms bound to macromolecules. The degree of covalent binding of the aflatoxins correlated with their cytotoxicity and genotoxicity at lower doses. After a 24-hr incubation, 12.5% of the dose of [14C]AFB1 was covalently bound to macromolecules compared to 1.5% of [3H]AFM1. Although AFM1 was less potent than AFB1 in cytotoxicity, DNA-repair and covalent-binding assays using primary cultures of hepatocytes, AFM1 was still active at relatively low doses and therefore is probably a potent hepatotoxin in vivo.

Isolation and culture of adult hepatocytes from liver biopsies.

Hepatocytes were isolated from liver biopsies of rats, guinea pigs, rabbits, dogs, and humans. The procedure is based on cannulation of large veins in the cut face of the biopsy, followed by collagenase perfusion. Yields averaged 19 x 10(6) viable hepatocytes/g liver. Viability averaged 84%, as determined by trypan blue dye exclusion. Cultures were prepared from the isolated hepatocytes and were found to be comparable in morphology and N-demethylase activity to hepatocyte cultures prepared by the in situ perfusion of the liver. The development of this method should facilitate comparative studies of the cytotoxicity, genotoxicity, and metabolism of foreign chemicals in primary hepatocyte cultures.