A novel inhibitor of beta-glucuronidase: L-aspartic acid.

Infants who consume casein hydrolysate formula have been shown to have lower neonatal jaundice levels than infants who consume routine formula or breast milk. Because casein hydrolysate has been shown to contain a beta-glucuronidase inhibitor, one possible mechanism to explain this finding is blockage of the enterohepatic circulation of bilirubin by a component of the formula. The aim of this research was to identify the source of the beta-glucuronidase inhibition in hydrolyzed casein. A beta-glucuronidase inhibition assay and measurements of physical and kinetic parameters were used to analyze the components of hydrolyzed casein and infant formulas. Kinetic studies used purified beta-glucuronidase. The L-aspartic acid in hydrolyzed casein accounts for the majority of the beta-glucuronidase inhibition present. Kinetic studies indicate a competitive inhibition mechanism. L-aspartic acid is a newly identified competitive inhibitor of beta-glucuronidase.

Use of maternal plasma level of zinc-coproporphyrin in the prediction of intrauterine passage of meconium: a pilot study.

OBJECTIVE: To evaluate the usefulness of maternal plasma zinc-coproporphyrin (ZCP) level as a marker for intrauterine passage of meconium. METHODS: A pilot study consisting of 10 pregnancies with meconium-stained amniotic fluid and 10 pregnancies with clear amniotic fluid was used. The corresponding plasma and amniotic fluid levels of ZCP were measured using spectrofluorometry. ZCP levels in plasma and amniotic fluid were compared between the two groups and the relation between plasma and amniotic fluid ZCP levels in the clear and meconium-stained groups was assessed using Spearman rank-order correlation. RESULTS: Mean amniotic fluid ZCP was significantly higher in the meconium-stained amniotic fluid as compared to the clear amniotic fluid group. Although mean plasma ZCP levels were higher in the meconium-stained amniotic fluid group, this difference was not statistically significant. There was no significant correlation between plasma ZCP levels and amniotic fluid ZCP, but we could categorize patients according to plasma ZCP levels into four categories with different risks for having meconium-stained amniotic fluid. CONCLUSIONS: Plasma ZCP might be a promising test for prediction of intrauterine passage of meconium in high-risk patients if confirmed by larger studies. The implications of this prediction on management remain unknown.

Inhibition of beta-glucuronidase by casein hydrolysate formula.

BACKGROUND: A casein hydrolysate infant formula has been shown to be associated with lower levels of neonatal jaundice than are standard infant formulas. Because beta-glucuronidase is related to neonatal jaundice, this study examined the effect of a casein hydrolysate formula on beta-glucuronidase. METHODS: Beta-glucuronidase activity was measured with or without added dietary components. The beta-glucuronidase sources used were meconium, breast milk, and the purified bovine liver enzyme. The dietary components assayed for their effect on beta-glucuronidase activity included casein hydrolysate formula (Nutramigen), whey-predominant formula (Enfamil), breast milk; enzymatically hydrolyzed casein, and other constituents of the casein hydrolysate formula. Stool samples of 6-day-old infants, who were exclusively fed one of the two formulas or breast milk, were also assayed for inhibition of beta-glucuronidase. RESULTS: Only Nutramigen, enzymatically hydrolyzed casein, and stool from Nutramigen-fed infants consistently demonstrated significant inhibition of beta-glucuronidase activity, ranging from 45% to 85% of that in controls. The inhibition of beta-glucuronidase in purified bovine liver demonstrates a dose response in a pH range from 4 to 7.3. CONCLUSIONS: Hydrolyzed casein contains a beta-glucuronidase inhibitor that, in casein hydrolysate-fed infants, persists after passage through the digestive tract. These data are consistent with the possibility that inhibition of beta-glucuronidase is a mechanism by which infants fed casein hydrolysate have lower jaundice levels than infants fed routine formulas or breast milk. Further study of this mechanism is needed.

Use of a low-speed, iso-density percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations.

A simple yet effective method (iso-density percoll centrifugation) has been developed for consistently preparing isolated rat liver parenchymal cells with over 98% initial viability. The method has been applied to cells isolated by a variety of collagenase digestion techniques. This procedure involves the low-speed centrifugation (50 X g) of the initial cell suspension through a percoll medium having a density of 1.06 g/ml and results in the separation of single and viable parenchymal cells from cell aggregates, debris, and nonparenchymal cells. The enriched parenchymal cells have been shown to be superior to untreated cells by a number of criteria including: preparation homogeneity, cell morphology, maintenance of cytochrome P-450, hormonal responsiveness (measured by the induction of tyrosine aminotransferase after treatment with glucagon or dexamethasone, or both), plasma membrane integrity (determined by both trypan blue exclusion and leakage of glutamic-oxaloacetic transaminase), and the DNA repair capability after treatment with benzo[a]pyrene or 2-acetylaminofluorene.

Delayed wasting syndrome and alterations of liver gluconeogenic enzymes in rats exposed to the TCDD congener 3,3', 4,4'-tetrachloroazoxybenzene.

A delayed wasting syndrome similar to that induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was observed in male Sprague-Dawley rats exposed to 3,3', 4,4'-tetrachloroazoxybenzene (TCAOB) and 3,3',4,4'-tetrachloroazobenzene (TCAB). After a slow growth period, all treatment animals (25 mg/kg, i.p., 2 doses per week) exhibited a starvation-like syndrome characterized by reduced food intake, dramatic loss of body weight and subsequent death. Although the growth of all major organs in the treatment animals was affected, the thymus appeared severely atrophied. The growth kinetics during the earlier phase were further analyzed using serially-killed rats receiving TCAOB. In addition, TCAOB was found to markedly depress the specific activity (mumol/min/g wet liver) of glucose-6-phosphatase, fructose-1,6-bisphosphatase, phosphoenolpyruvate carboxykinase, and pyruvate kinase in the liver. Significant changes in the levels of cytochrome P-450, glutamic-pyruvic transaminase and malic enzyme in the liver were also observed.

Induction of DNA damage and repair synthesis in freshly isolated rat hepatocytes by the proallatocidin precocene II.

The proallatocidin precocene II (6,7-dimethoxy-2,2-dimethyl-2H-benzo-[b]pyran) has previously been shown to induce centrolobular liver necrosis. Here we have examined the ability of precocene II to produce DNA damage in suspensions of freshly isolated rat hepatocytes using the alkaline elution technique with N-methyl-N'-nitro-N-nitrosoguanidine as a positive control. At concentrations (10(-4)-10(-5) M) which did not induce cytotoxicity as judged by the leakage of glutamic-oxaloacetic transaminase, precocene II was capable of producing DNA single-strand breaks. In addition, a dose-dependent DNA repair synthesis (unscheduled DNA synthesis, UDS) was detected in hepatocytes exposed to precocene II. The induction of UDS was measured by incorporation of [3H]thymidine into purified hepatic DNA via a membrane filter retention method and liquid scintillation counting. Hence, results obtained in the present study indicate the potential genotoxicity of precocene II and the utility of DNA damage and repair assays in genetic toxicology.

Immunosuppression in weanling and adult Sprague-Dawley rats induced by acute exposure to 3,3',4,4'-tetrachloroazoxybenzene.

The acute immunomodulatory effects of the environmental and occupational contaminant, 3,3',4,4'-tetrachloroazoxybenzene (TCAOB), were investigated on selected immune parameters in weanling and adult Sprague-Dawley rats. Significant immunotoxic effects were found 17 days after 4 doses of 25 mg/kg TCAOB, administered i.p. The main non-immune toxic effects were decreased body, kidney, heart and testis weights, and a simultaneous increase in liver weight. The immune parameters showing significant suppression were: thymic weight, splenic plaque forming cell populations and function, pertioneal macrophage chemiluminescence, and bone marrow cellularity. Weanling animals were affected by TCAOB to a greater extent than adults on both the multiple and single dose regimens. The immunotoxic effects were found to be qualitatively similar to those of its isosteric analog, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The results demonstrate that TCAOB is a very potent immunotoxic compound and may have long-term effects after a single exposure. This study is the first investigation into the effect of TCAOB on immune functions.

A rapid and simple method to quantitate chemically induced unscheduled DNA synthesis in freshly isolated rat hepatocytes facilitated by DNA retention of membrane filters.

The filter elution method used for the detection of DNA strand breaks has been modified to quantitate chemically induced DNA repair which is measured as unscheduled DNA synthesis (UDS) in suspension of freshly isolated rat hepatocytes. Our method is based on DNA purification by retention on polyvinyl chloride filters, and is capable of handling a large number of samples simultaneously. By using the present assay system, positive dose-dependent UDS data was obtained on the following carcinogens: aflatoxin B1, 2-acetylaminofluorene, 4-aminobiphenyl, 2-aminofluorene, methyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, and 4-nitroquinoline-1-oxide. In contrast, non-carcinogenic biphenyl, fluorene, and sodium ascorbate did not elicit any detectable levels of UDS at all concentrations tested. Thus, UDS as measured by the present filter retention method may serve as an efficient and reliable means of screening chemical mutagens/carcinogens.

DNA damage of mammalian cells by the beef extract mutagen 2-amino-3-methylimidazo[4,5-f]quinoline.

The beef-extract mutagen, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), was shown, by alkaline elution procedures, to induce DNA damage in radiation-induced mouse leukaemia cells. The effect, which was dose related, occurred in incubations containing S-9 mix derived from polychlorinated biphenyl-induced rat liver but not in the absence of this metabolic activation system. An increased alkaline elution of DNA was also observed following IQ addition to cultures of hepatocytes from 3,3',4,4'-tetrachloroazobenzene-induced rat liver, and the DNA damage was again dose related. IQ has thus been shown to be genotoxic to mammalian cells in the presence of an effective activation system.

Embryotoxicity of 3,3',4,4'-tetrachloroazobenzene and 3,3',4,4'-tetrachloroazoxybenzene in the chick embryo.

The toxicity of 3,3',4,4'-tetrachloroazobenzene (TCAB) and 3,3',4,4'-tetrachloroazoxybenzene (TCAOB) to chick embryos was examined. TCAB or TCAOB was dissolved in corn oil and injected into the air cell of fertile chicken eggs. The time of injection had a major effect on embryo mortality as eggs injected with TCAB or TCAOB on the fourth day of incubation had a higher incidence of embryo mortality than eggs injected on days 11-13. Both TCAB and TCAOB were more toxic than all other chemicals that have been tested in the chick embryo with the exception of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Comparing the potency of the two compounds, TCAOB was more potent than TCAB in the chick embryo with an estimated LD50 of 12 ng and 44 ng respectively. Rump edema was the major abnormality observed in embryos treated with either TCAB or TCAOB. Other malformations included altered feather pattern and lack of down, hemorrhage, external viscera, reduced body size, failure to withdraw the yolk sac, beak malformation, dilation of blood vessels, and monomicropthalmia. The results of this investigation suggest that both TCAB and TCAOB are teratogenic in the chick embryo.

Thymic atrophy induced by acute exposure of 3,3',4,4'-tetrachloroazobenzene and 3,3',4,4'-tetrachloroazoxybenzene in rats.

The effects of 3,3',4,4'-tetrachloroazobenzene (TCAB) and 3,3',4,4'-tetrachloroazoxybenzene (TCAOB) on lymphoid organs in male Sprague-Dawley rats were investigated in various acute exposure studies. Significant thymic atrophy was observed in rats 10 days after the i.p. administration of either compound (on days 1 and 5) at a dose of 25 mg/kg. When 8-week-old animals were studied, the relative thymus weight was reduced to 69% and 49% of the control value by the respective treatment of TCAB and TCAOB. In 2 groups of weanling rats the same dosage of TCAOB was able to reduce the relative thymus weight to 31% and 38% of the comparable control animals. In addition, TCAOB causes a decrease in the body weight gain and a decrease in the weights of major organs in the weanling animals. This toxic response cannot be explained solely on the basis of decreased food intake since qualitatively the same results were observed in a pair-feeding experiment. The involvement of glucocorticoid hormones was rejected as the underlying mechanism since adrenalectomy was found to provide no protection towards the degenerative effect seen upon the lymphoid tissues. This investigation constitutes the first study concerning the effects of these 2 environmental and occupational toxicants on lymphoid organs.

Metabolism studies of 3,3',4,4'-tetrachloroazobenzene. I. In vitro metabolic pathways with rat liver microsomes.

The metabolism of 3,3',4,4'-tetrachloroazobenzene (TCAB), an important environmental and occupational toxicant, by rat liver microsomes has been examined in a NADPH-generating system. The metabolic pathways were delineated by the combined use of high pressure liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). The rate of [14C]TCAB metabolism using induced microsomes was found to be 381 +/- 59 pmol/min/mg microsomal protein. Approximately 13% of the radioactivity from the [14C]TCAB substrate was covalently bound to the macromolecular pellet at the end of a 2-h incubation period. In addition, three distinct TCAB metabolites were isolated from the organic extracts and subsequently identified. Experiments with carbon monoxide, 2-diethylaminoethyl 2,2-diphenylvalerate hydrochloride (SKF 525-A), and control (uninduced) microsomes indicated the participation of the cytochrome P-450-dependent monooxygenases. The biological significance of both oxidative and reductive metabolic pathways were discussed. It was suggested that the generation of a reactive arene oxide intermediate mediated by oxidative enzymes may be crucial for some of the TCAB toxic effects observed in rodent tissues.

General health effects of prolonged exposure to 3,3',4,4'-tetrachloroazobenzene and 3,3',4,4'-tetrachloroazoxybenzene in rats.

3,3',4,4'-Tetrachloroazobenzene (TCAB) and 3,3',4',4'-tetrachloroazoxybenzene (TCAOB) are formed as contaminants during the industrial manufacturing of chloroanilide herbicides. Changes in the general body health status were evaluated in rats fed an experimental diet containing 100 ppm of TCAB or TCAOB for 120 days. A significant difference in body weight between experimental and control animals was observed. Of all major organs examined at necropsy, liver, spleen and testis weight were affected. Induction of hepatic drug-metabolizing enzymes was observed. Hematological studies indicated a definite reduction in the number of erythrocytes among TCAOB treated rats. Various parameters of serum clinical chemistry were subsequently determined. The total lipids and glutamic-oxalacetic transaminase in exposed animals were found to be elevated. Results obtained in this study coupled with our earlier findings on carcinogenicity indicate that occupational exposure to TCAB and TCAOB may cause adverse human health effects.

Induction of hepatic microsomal cytochrome P-448 by 3,3',4,4'-tetrachloroazobenzene and the corresponding azoxy and hydrazo analogs.

3,3',4,4'-Tetrachlorazobenzene (TCAB) has been found to pose occupational health hazards among chemical workers as a consequence of its contamination in commercially important herbicides. The induction of hepatic microsomal cytochrome levels in male rats treated with TCAB or its azoxy and hydrazo analogs were examined in several short-term experiments. Five daily consecutive administrations of TCAB or 3,3',4,4'-tetrachloroazoxybenzene (TCAOB) at doses of 25 mg/kg/day yielded maximal stimulation of cytochrome levels. Both compounds induced cytochrome P-450 with absorption maxima (reduced, CO complex) at 448 nm to 2.7 times control levels. In contrast, cytochrome P-448 levels were enhanced less than twofold relative to controls in animals treated with 3,3',4,4'-tetarchlorohydrazobenzene (TCHB) under identical conditions. Cytochrome inductions arising from these chemicals are dose dependent. Parallel increase of the liver to body weight ratios was also observed in these studies. Further experiments using different regimens indicated TCAOB as being the most potent and persistent inducer among these three analogs. A twofold enhancement of cytochome P-448 levels was observed 14 days after a single ip injection of TCAOB at doses as low as 10 mg/kg.

Induction of unscheduled DNA synthesis in suspensions of rat hepatocytes by an environmental toxicant, 3,3'4,4'-tetrachloroazobenzene.

Unscheduled DNA synthesis was induced by 3,3'4,4'-tetrachloroazobenzene (TCAB)) in freshly isolated suspensions of rat hepatocytes. A dose-dependent response was demonstrated. Hepatocellular DNA was obtained after the chloroform-isoamyl alchohol-phenol extraction of the isolated nuclei. The induction of unscheduled DNA synthesis was measured by the incorporation of [3H]-thymidine in the presence of hydroxyurea as determined by the scintillation counting assay. DNA repair data obtained in this study on benzo[a]pyrene and methyl methanesulfonate are comparable to a previous report using primary cultures of hepatocytes and cesium chloride gradients. Hence, the present method offers promise as a rapid and sensitive screen for chemical carcinogens.

Inhibition of cell-free protein synthesis by analogues of aurintricarboxylic acid.

Aurintricarboxylic acid is a potent inhibitor of cell-free protein synthesis by the post-mitochondrial supernatant of chick brain and the translation of mRNA by wheat germ lysate. Comparison of commercially available and chemically synthesized analogues of aurintricarboxylic acid indicates that the unique aurin triphenyl methane ring system and the carboxylic acid groups are both necessary for inhibition of cell-free protein synthesis in both systems.