Theodore M. Farber

Effect of polychlorinated biphenyls on hepatic microsomal enzymes in the rat

Abstract Polychlorinated biphenyls (PCB) are widely used industrial synergists and stabilizers that have been detected in various animal systems throughout the world. Although these chemicals possess a low acute toxicity, recent indications are that they may possess a high chronic toxicity. To evaluate further the toxicity of PCB mixtures, rats were fed diets containing 4 different Aroclors varying in chlorine content from 42 to 60% for 4 wk at levels of 0.5, 5, 50, and 500 ppm. The influence of PCB was studied by determining liver weights, hepatic triglyceride concentration and glucose-6-phosphatase activity, microsomal enzyme activities (nitroreduction, demethylation and hydroxylation) and microsomal cytochrome P-450 content. Liver-to-body weight ratios and cytochrome P-450 content were significantly increased at 50 and 500 ppm for all PCB mixtures; the increase in cytochrome P-450 was somewhat greater with increasing chlorine content. Significant increases in liver triglycerides were seen at 500 ppm, with a maximum effect occurring at approximately 50% chlorine content. A dose-related increase in demethylase activity was observed, with a 2.5- to 3-fold increase over control values at 500 ppm for all mixtures. Effects were maximum at approximately 50% chlorine content. A dose-related increase in pentobarbital hydroxylation was seen, and the induction of hydroxylation increased with increasing chlorine content. Nitroreductase activity was markedly stimulated with increasing doses of PCB, and induction was seen at levels as low as 0.5 ppm for all mixtures. The maximal effect appears to be at levels higher than 500 ppm. The highest levels of activity were seen with 60% chlorination.

Biochemical and cytogenetic effects in rats caused by short‐term ingestion of aroclor 1254 or firemaster BP6

Male rats were fed either 0, 5, 50, or 500 ppm Aroclor 1254 (ARO) or Firemaster BP6 (FM) mixed in ground rat chow for 2, 3, or 5 wk. The relative biological response to the two chemicals sometimes varied with dose level. For example, for some parameters FM caused a greater effect than ARO at the 5 and 50 ppm levels, but not at the 500 ppm level, where, presumably, the maximum response was attained. The experimental design did not permit a totally unambiguous comparison of all the responses to ARO and FM. Some apparent differences in biochemical responses determined at sacrifice may have been partly due to the fact that, for the 3 wk study, rats were exposed to FM 48 hr longer than to ARO, and thus the comparison of ARO and FM toxiclties should be considered tentative. The following evaluations were made, based on the assumption that this 2 day difference had no effect. Relative kidney and testis weights, hematology, SGPT, SGOT, BUN, plasma corticosterone, rate of liver protein synthesis per gram of tissue...

Endothelial lesions in the aorta of egg yolk-fed miniature swine: A study by scanning and transmission electron microscopy

Abstract Evidence has been accumulating which suggests that atherosclerotic lesions are preceded by endothelial cell damage. Ten miniature swine (FDA, Beltsville, Md.) were maintained on an egg yolk diet (six animals) or on a control diet (four animals) beginning at 5 weeks of age. All animals were sacrificed at 52 weeks of age by intracardiac perfusion of glutaraldehyde. Segments of the aortae were excised and prepared for scanning (SEM) and transmission (TEM) electron microscopy. SEM examination of the aortae of egg yolk-fed animals revealed crater-like defects in the endothelial surface associated with marked cellular deformation and disruption. With TEM there were areas of focal disruption of the luminal plasma membrane and exposure of subendothelial tissues. These alterations were found most commonly on or near the leading or following lip or lateral to branch orifices. Similar endothelial lesions (crater-like defects) have been found in monkey and rabbit arteries following ischemia and in rabbit arteries following cholesterol feeding, epinephrine injection, mechanical trauma, and delayed fixation. It is believed that these alterations are a nonspecific reaction of endothelial cells to injury and may represent the initial morphological abnormality in the pathogenesis of atherosclerosis.

Comparative distribution, excretion and metabolism of di-(2-ethylhexyl) phthalate in rats, dogs and miniature pigs.

Abstract Di-(2-ethylhexyl phthalate (DEHP) was administered in the diet to male Sprague-Dawley rats, beagle dogs and miniature swine of the Hormel strain in doses of 50 mg/kg/day for 21–28 days before administration of a single dose of [14C]DEHP at 50 mg/kg. The animals were then killed at various times, and tissues, organs, urine and faeces were analysed for distribution of radioactivity. Approximately 84% of the [14C]DEHP radioactivity was excreted in the urine and faeces of rats during the first 24 hr; in dogs and pigs, excretion during this time was 67 and 37%, respectively. Elimination of 14C was rapid in rats, slightly prolonged in dogs and least rapid in pigs; excretion in all three species was virtually complete in 4 days. Faecal excretion (75%) predominated in dogs, and urinary excretion (79%) predominated in pigs. Thin-layer chromatography showed the presence of at least four radioactive substances in rat urine, three in dog urine and five in pig urine. No more than a trace of unmetabolized DEHP was found in the urine of rats, dogs or pigs. In all three species, bile contained some metabolites that differed from those in urine, although some metabolites appeared to be common to both. Bile from rats and dogs contained a substance that, when hydrolysed under slightly acidic conditions, yielded a substance that migrated like mono-(2-ethylhexyl) phthalate in the chromatographic system used.

Elevation of serum cholesterol and increased fatty streaking in egg yolk:lard fed castrated miniature pigs.

The effects of feeding an experimental diet containing 16.25% (w/w) dry powdered egg yolk and 30.8% total fat (20% from lard) were compared with an isocaloric amount of control (stock) diet in castrated (N = 11 each) and sham-operated (N = 8 each) male miniature pigs of Hormel origin. Piglets were castrated at 2 weeks of age. The experimental diet was fed from 9 weeks of age and serum lipids (cholesterol and triglycerides) and plasma testosterone were determined at 5-week intervals up to 52 weeks of age. At 52 weeks of age, the animals were sacrificed and the aortas evaluated for fatty streaking after Sudan IV staining. Castration was accompanied by significantly (P <= 0.02) elevated serum cholesterol (castrated 84 ± 4 mgm/dl vs. sham, 67 ± 5 mgm/dl; mean ± SEM) within 5 weeks after feeding the stock diet and throughout the duration of the expriment. Except for the initial cholesterol concentration (castrated, 118 mgm/dl vs. sham, 88 mgm/dl), both castrated and sham-operated animals on egg yolk had significantly higher (P <= 0.05) serum cholesterol than their littermate controls. At 52 weeks of age, the serum cholesterol of sham-operated and castrated animals on experimental diet were 152 ± 29 and 292 ± 41 mgm/dl as compared with 68 ± 5 and 96 ± 4 mgm/dl for their littermates on stock diet respectively. No correlation was found between the concentrations of plasma testosterone and serum cholesterol. Consistent with the elevation of serum cholesterol, the castrated animals on egg yolk diet had significantly greater area of the aorta covered with fatty streaking than did the sham-operated group. These data show that castration and feeding of egg yolk: lard diet resulted in hypercholesterolemia and increased fatty streaking.

Distribution and excretion of two phthalate esters in rats, dogs and miniature pigs

Abstract Diisooctyl phthalate (DIOP) or butylglycolylbutyl phthalate (BGBP) was administered in the diet to male Sprague-Dawley rats, beagle dogs and miniature pigs in a dose of 50mg/kg for 21-28 days before oral administration of a single dose of the same compound labelled with 14 C in the carbonyl group. The animals were killed and tissues, faeces and urine were analysed for 14 C at various times after the dose of [ 14 C]phthalate was administered. In the rat, approximately half of the 14 C activity from labelled DIOP was excreted in the urine and half in the faeces, while radioactivity from this phthalate appeared predominantly in the faeces in dogs (69-80%) and in the urine in pigs (65-86%). The BGBP label was excreted mainly in the urine in rats (77-83%), dogs (72-75%) and pigs (68-89%). Extraction of the tissues for metabolites indicated that DIOP was less readily metabolized than BGBP by all three species. The nature of the substituent on the phthalate molecule appears to influence the excretion of the phthalate-ester metabolites.

Drug-induced changes in serum alkaline phosphatase and alanine aminotransferase activities not related to hepatic injuries.

Changes in serum alkaline phosphatase (SAP) and alanine aminotransferase activities are of clinical diagnostic significance in determining hepatic injuries. Drugs, however, can alter the activities of these enzymes by various mechanisms. Phenobarbital increases SAP activity in dogs as a result of hepatic microsomal induction (Litchfield and Corning, 1971). We examined the effect of related enzyme inducers. Beagle dogs (2/sex/group) were dosed orally with phenobarbital or diphenylhydantoin at 40 mg/kg or primidone at 40 to 80 mg/kg for 7 weeks. Only phenobarbital increased SAP; bromsulfophthalein retention was not affected. Plasma antipyrine half-life decreased and liver cytochrome P-450 content increased in each group compared with values from a control group. No histological changes of the liver were seen. Data indicate the specificity of phenobarbital. Some drugs decrease serum enzyme activities, e.g., alanine aminotransferase activity. Cefazolin was given s.c. to rats at 2 g/kg/day on days 1–18 and at 0.5 g/kg/day on days 19–32. Isoniazid was given orally at 20 mg/kg/day for 33 days to a similar group. Half of each group received pyridoxal HCl s.c. at 0.2 mg/ kg/day on days 1–18 and at 2 mg/kg/day on days 19–46. Serum alanine aminotransferase decreased in each group, but returned to normal on day 32 in rats dosed with isoniazid-pyridoxal combination. Isoniazid is known to interfere with alanine aminotransferase activity by decreasing the availability of this coenzyme. The mechanism of the cefazolin effect has not been elucidated.

The toxicity of brominated sesame oil and brominated soybean oil in miniature swine

Miniature swine were fed brominated sesame oil at dietary levels of 0, 5, 25, 50 or 500 mg/kg of body weight for 17 weeks and brominated soybean oil at levels of 0, 5, 50 or 500 mg/kg of body weight for 28 weeks. Growth rate and food intake were decreased only at the high dose level in the brominated sesame oil study. In both studies, signs of lethargy and ataxia occurred in pigs fed the highest dose, and were probably due to a dose-related increase in serum bromine concentrations. Marked elevations in lactic dehydrogenase (LDH), serum glutamic-oxalacetic transaminase (SGOT) and serum glutamicpyruvic transaminase (SGPT) values were seen at the highest dose level with both substances and these enzyme activities were increased at the 50 mg/kg dose level in the brominated sesame oil study. Histopathologic lesions were confined to animals given the highest dose level of either oil. Marked fatty degeneration of the hepatic plate cells and renal tubular epithelial cells were seen in both studies. In the brominated sesame oil study, neutral fat was moderately increased in the myocardium of the pigs fed 500 mg/kg. However, marked diffuse accumulation of LDH, marked diffuse fatty degeneration and focal degeneration, and/or necrosis of individual or small groups of cardiac muscle fibers were seen in the group fed brominated soybean oil at 500 mg/kg. A moderate to marked testicular atrophy was also observed in this group. A dose-related accumulation of total and hexane-soluble bromine was observed in all tissues examined in both studies; the highest concentrations occurred in adipose tissue of the pigs given the highest dose level. Kidneys, livers, hearts and thyroids of these groups also contained large amounts of bromine. In pigs given the 50 mg/kg dose level, total and hexane-soluble bromine concentrations were higher in the brominated sesame oil study than in the longer brominated soybean oil study and may be responsible for the elevations in LDH, SGPT and SGOT activities in this group.

The effect of lindane and phenobarbital on microsomal enzyme induction in dogs and miniature swine.

Abstract The enzymic activities of 9000g supernatant fractions obtained from the livers of normal beagle dogs and miniature swine and of dogs and swine treated with lindane and phenobarbital are presented. No significant sex differences in activities were noted in control dogs. Preparations from dogs of both sexes fed lindane at 7.5 and 15.0 mg/kg exhibited a significant loss in codeine demethylase activity while nitroreductase activity was increased twofold. Phenobarbital pretreatment of dogs and swine stimulated all microsomal systems tested. Addition of lindane to the diet of these induced dogs caused a significant and rapid decline in the values after only two feedings. Enzymes induced to a greater extent in dogs by phenobarbital were inhibited to a greater extent by lindane; i.e., as a class, demethylases were affected by both lindane and phenobarbital to a greater extent than aromatic hydroxylases. In contrast, administration of lindane to phenobarbitaltreated swine caused an actual further increase in demethylase activities. A decrease in the induced state was observed in aromatic hydroxylation, azo-reduction, and nitro-reduction reactions, although the activities of these enzymes were still higher than the values from control swine.

Instrumental neutron activation analysis for bromine in pig tissues

Abstract Bromine was determined by instrumental neutron activation analysis in tissues and organs of pigs as an indicator of brominated vegetable oil residues. The bromine content was found to be dose-related. An advantage of this technique is that the sample was not treated chemically. The analyses were done with the aid of an automatic sample changer and computer reduction of the data. Rapid and reliable analyses were obtained at the p.p.m. level.