James L. Byard

Maintenance of cytochrome P-450 and metabolism of aflatoxin B1 in primary hepatocyte cultures

The cytochrome P-450 content of primary hepatocyte cultures was maintained at levels close to those found in vivo by using a defined medium containing testosterone, thyroxine, hydrocortisone, estradiol, glucagon, insulin, linoleic acid and oleic acid. Using these cultures, [14C]aflatoxin B1, a potent liver carcinogen, was metabolized primarily to water-soluble metabolites. In agreement with in vivo results, aflatoxin M1 was the only nonpolar metabolite detected. In addition, a significant portion of radioactivity was covalently bound to cell constituents. These results suggest that primary hepatocyte cultures may be a good model of the liver for studying the metabolism and mechanism of action of toxic chemicals.

Isolation and culture of adult hepatocytes from liver biopsies

SummaryHepatocytes 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 19×106 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 andn-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.

Butylate hydroxytoluene pretreatment protects against cytotoxicity and reduces covalent binding of aflatoxin B1 in primary hepatocyte cultures

Primary cultures of adult rat hepatocytes were used to characterize the effect of butylated hydroxytoluene (BHT) pretreatment on the metabolic disposition and cytotoxicity of a single dose of aflatoxin B1 (AFB1). Four male Sprague-Dawley rats were fed a control diet, and five were fed a diet containing 0.5% BHT. After 10 days, hepatocytes were prepared and cultured in chemically defined, hormone-supplemented medium. After 20–22 hr in culture, 120–150 ng of [14C]aflatoxin B1 was added to dishes containing 2.5 × 106 cells. By 10 hr, control cells had converted 59% of the AFB1 to aqueous metabolites, while 15.5% was bound covalently. During the same interval, cells from BHT-fed rats produced 69% aqueous metabolites, and only 6.6% was bound covalently. The rate of AFB1 disappearance in the two groups was not statistically different. AFB1 produced marked cytotoxicity in hepatocyte cultures from control rats but had no apparent toxic effect on hepatocyte cultures from BHT-pretreated rats, as indicated by light microscopic examination and release of lactate dehydrogenase into the medium. These results suggest that reduction of cytotoxicity by BHT was associated with increased output of nontoxic, water-soluble metabolites and decreased binding of metabolites to macromolecules. These results also indicate that BHT may protect against the acute toxicity and carcinogenicity of aflatoxin B1 in vivo.

Primary cultures of adult mouse and rat hepatocytes for studying the metabolism of foreign chemicals

Abstract A primary hepatocyte culture was developed as a model system to investigate the metabolism of foreign chemicals. Hepatocytes were prepared from adult male Charles River CD-1 mice and adult male Sprague-Dawley rats by in situ pre-perfusion of the liver with ethyleneglycol-bis-(β-amino-ethyl ether) N , N ′-tetra-acetic acid (EGTA) followed by perfusion with calcium and collagenase. The digested liver was dispersed, and hepatocytes were isolated by filtration and differential centrifugation yielding 10 8 hepatocytes per mouse liver and 5 × 10 8 hepatocytes per rat liver. More than 90 per cent of the hepatocytes excluded trypan blue. Hepatocytes were prepared aseptically, plated on tissue culture dishes coated with rodent tail collagen (2.5 × 10 6 cells/60 mm dish), and cultured in serum-free modified Waymouths medium. Within 4hr the hepatocytes attached to the collagen, and by 24 hr they had flattened and formed a monolayer. A non-metabolizable alanine analog, α-aminoisobutyric acid, accumulated in mouse hepatocytes with peak incorporation occurring at 24 hr. Cultured mouse and rat hepatocytes were able to N -demethylate para-chloro- N -methylaniline (PCMA). An NADPH-generating system stimulated N -demethylation 2.75-fold in freshly isolated mouse hepatocytes, but did not stimulate metabolism in cultured mouse hepatocytes. SKF 525-A inhibited PCMA N -demethylation in cultured mouse hepatocytes with an I 50 of 3.75 × 10 −5 M. Hormonal supplementation of the culture medium stimulated PCMA metabolism measured in 24- and 48-hr cultures. These studies demonstrate the utility of rodent hepatocyte cultures as models of hepatic metabolism of foreign chemicals.

Metabolism, cytotoxicity, and genotoxicity of the pyrrolizidine alkaloid senecionine in primary cultures of rat hepatocytes

Abstract Senecionine, a pyrrolizidine alkaloid isolated from Senecio vulgaris, was a potent toxicant to primary cultures of adult male rat hepatocytes. A nontoxic dose of [14C]senecionine (1.9–2.4 nmol/106 cells) was rapidly taken up from the culture medium by hepatocytes and converted to more polar metabolites recovered in the culture medium. Concomitantly some metabolites formed stable covalent bonds with cellular macromolecules. After a 24-hr incubation, 88% of the dose was water soluble while only 10% could be extracted with chloroform; 1.3% of the dose was bound to trichloroacetic acid-precipitated material and could not be extracted with organic solvents. Senecionine was a potent cytotoxin as evidenced by the leakage of lactate dehydrogenase from the hepatocytes into the surrounding culture medium and the loss of cells from the collagen substratum. A linear dose-response relationship for cytotoxicity was obtained for doses ranging from 2.6 to 128 nmol per 106 cells. Senecionine was also genotoxic as suggested by the evidence of covalent binding and the stimulation of DNA repair. The lowest dose that stimulated DNA repair was 16 nmol/106 cells. Based on these data, one would predict that senecionine is not only a potent hepatotoxin but is also likely to be carcinogenic.

Metabolism of aflatoxin B1 in cultured mouse hepatocytes: Comparison with rat and effects of cyclohexene oxide and diethyl maleate☆

The metabolism of aflatoxin B1 was studied in cultured hepatocytes from adult male mice. After a 10-hr incubation, 89% of the dose of [14C]aflatoxin B1 was converted to aqueous metabolites, while 0.77% was covalently bound to cellular macromolecules. In contrast, it has been previously shown that hepatocytes cultured from the rat and incubated with aflatoxin B1 form 57.2% aqueous metabolites and covalently bind 12.2% to cellular macromolecules. The nearly 16-fold greater covalent binding in rat hepatocytes correlates with the greater susceptibility of the rat to the hepatocarcinogenic effect of aflatoxin B1. Diethyl maleate and cyclohexene oxide each was shown to significantly increase the amount of covalent binding in hepatocytes from both species. At 10−3 m, diethyl maleate and cyclohexene oxide, respectively, produced 805 and 61% increase in covalent binding in mouse hepatocytes, suggesting that both diol formation and glutathione conjugation are important detoxification pathways for aflatoxin B1. Both pathways are apparently more active in mouse hepatocytes than in rat hepatocytes.

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.

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.

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.

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.