M. Wilson Tabor

Intervention in free radical mediated hepatotoxicity and lipid peroxidation by indole-3-carbinol

The cytoprotective effect of the natural dietary constituent indole-3-carbinol (I-3-C) on carbon tetrachloride (CCl4) mediated hepatotoxicity in mice was examined. I-3-C pretreatment by gavage 1 hr prior to intraperitoneal injection of CCl4 produced a 63% decrease in CCl4-mediated centrolobular necrosis and a related 60% decrease in plasma alanine aminotransferase activity (a marker of liver necrosis). Since the toxicological effects of CCl4 are mediated by radical species generated during reductive metabolism by cytochrome P-450, we examined the potential ability of I-3-C to scavenge reactive radicals. Three systems were used to evaluate the ability of I-3-C to intervene in free radical mediated lipid peroxidation. These systems consisted of the following: (1) phospholipid dissolved in chlorobenzene, with peroxidation initiated by the thermal and photo decomposition of azobisisobutyronitrile (AIBN); (2) sonicated phospholipid vesicles in phosphate buffer (pH 7.4), with peroxidation initiated by ferrous/ascorbate; and (3) mouse liver microsomes containing an NADPH-regenerating system, with peroxidation initiated with CCl4. Lipid peroxidation was measured in these three systems as thiobarbiturate-reacting material. In the AIBN and ferrous/ascorbate systems, I-3-C inhibited lipid peroxidation, with greater inhibition under conditions of low rates of free radical generation. I-3-C was not as effective an antioxidant as butylated hydroxytoluene (BHT) or tocopherol, but it inhibited peroxidation in a dose-response manner. I-3-C was most effective as a radical scavenger in the microsomal CCl4-initiated system by inhibiting lipid peroxidation in a dose-dependent fashion, with 50% inhibition at 35-40 microM I-3-C. This concentration is about one-third of the concentration of I-3-C achieved in liver after treatment of mice by gavage with 50 mg I-3-C/kg body weight. These data suggest that I-3-C may be a natural antioxidant in the human diet and, as such, may intervene in toxicological or carcinogenic processes that are mediated by radical mechanisms.

Mechanisms of protection from menadione toxicity by 5,10-dihydroindeno[1,2,-b]indole in a sensitive and resistant mouse hepatocyte line

Established cell lines derived from newborn livers of c14CoS/c14CoS and cch/cch mice have been shown to be genetically resistant (14CoS/14CoS cells) or susceptible (ch/ch cells) to menadione toxicity. These differences are due in part to relatively higher levels of reduced glutathione (GSH) and NAD(P)H:menadione oxidoreductase (NMO1) activity in the 14CoS/14CoS cells. The indolic membrane-stabilizing antioxidant 5,10-dihydroindeno[1,2-b]indole (DHII) was shown previously to protect against various hepatotoxicants in vivo and in primary rat hepatocytes. This report describes how the 14CoS/14CoS and ch/ch cell lines provide a valuable experimental system to distinguish the mechanism of chemoprotection by DHII from menadione toxicity. The addition of 25 microM DHII produced a time-dependent decrease in menadione-mediated cell death in 14CoS/14CoS cells, with little effect on ch/ch cell viability. The maximum protective effect occurred at 24 hr, although the concentration of DHII remained constant for 48 hr. The protective effect of DHII correlated with enhanced glutathione levels (234% increase at 24hr), as well as induction of four enzymes involved in the detoxification and excretion of menadione: NAD(P)H:menadione oxidoreductase (NMO1, quinone reductase), glutathione reductase, glutathione transferase (GST1A1), and UDP glucuronosyltransferase (UGT1*06), with 24-hr maximum induction of 707, 201, 171 and 198%, respectively. Other biotransformation enzymes not directly involved in menadione metabolism (glutathione peroxidase, cytochromes P4501A1 and P4501A2, copper-, zinc-dependent superoxide dismutase, and NADPH cytochrome c oxidoreductase) were not induced by DHII. Menadione-stimulated superoxide production was inhibited 50% by DHII only in 14CoS/14CoS cells, and the inhibition required 24-hr preincubation. Pretreatment with DHII also protected both cell types against the menadione-mediated depletion of GSH, and the increase in percent (oxidized glutathione GSSG), an indicator of oxidative stress. These results suggest that DHII does not protect against menadione toxicity by virtue of its antioxidant or membrane-stabilizing properties. Rather, it acts by inducing a protective enzyme profile that migates redox cycling and facilitates excretion of menadione.

Exogenous Iron Overload in Perinatal Hemochromatosis: A Case Report

We report an infant with fatal neonatal liver disease in whom efforts to correct an associated unremitting anemia resulted in massive exogenous iron overload that was expressed as perinatal hemochromatosis (PH). Levels of iron and copper were elevated in multiple tissues. Echovirus subtype 9, recovered from the urine at age 3 weeks, may have been etiologic in the liver failure. PH is best viewed as a definable phenotype with an undefined genetic and/or environmental basis that emerges only in the context of severe perinatal liver disease. The absence of hemosiderin in splenic and bone marrow reticuloendothelial (RE) cells of our patient suggests an important role for RE cell dysfunction.

Antioxidation Potential of Indole Compounds — Structure Activity Studies

Many indole compounds, including dietary indoles such as indole-3-carbinol [I-3- C], have been shown to protect against chemical carcinogenesis (Wattenberg 1983) and chemically induced hepatotoxicity (Shertzer et al. 1987a, 1987b, 1988a). Such protection may be due to the ability of I-3-C to scavenge biologically reactive electrophiles (Shertzer et al. 1987b, 1988b) and free radicals (Shertzer et al. 1987a; Shertzer et al. 1988a). To develop better chemoprotective indoles with lower intrinsic toxicity, 28 structurally related indoles were investigated relative to antioxidant efficacies and physicochemical properties (Log P and electronic effects).

Changes in amniotic fluid glucose, β-hydroxy-butyrate, glycerol, and lactate concentration in diabetic pregnancy

Amniotic fluid glucose, beta OH butyrate, glycerol, and lactate concentrations were measured in 75 samples collected in the third trimester of pregnancy from 50 diabetic patients, all but four of whom required insulin. Increases in maternal fasting plasma sugar were accompanied by corresponding increases in amniotic fluid glucose and on occasion increases in amniotic fluid beta OH butyrate. These data correspond to previous reports of placental glucose transfer and in addition, provide statistically significant evidence of placental betaOH butyrate transfer since the hyperglycemic, hyperinsulinemic fetus of a diabetic mother would be a poor primary source for ketogenesis. Relatively poor correlation of elevated fluid levels of these solutes to fetal outcome probably reflects a low incidence of maternal hyperglycemia, ketogenesis. Relatively poor correlation of elevated fluid levels of these solutes to fetal outcome probably reflects a low incidence of maternal hyperglycemia, ketoacidosis, and over-all reduced neonatal morbidity-mortality rates in this group of metabolically well-controlled, predominantly insulin-requiring diabetic patients managed in a regional high-risk perinatal center.

Indole-3-carbinol Inhibits Lipid Peroxidation in Cell-Free Systems

Free radicals mediate toxicological and carcinogenic responses of tissues to many chemicals. Cellular defenses against radical mediated damage utilize endogenous substances such as tocopherol, ascorbate and GSH. Here we report a new antioxidant, indole-3-carbinol (I-3-C), a natural constituent of human diet. In chlorobenzene containing soy phospholipids, lipid oxidation was initiated with azobisisobutyronitrile; I-3-C inhibited formation of thiobarbituric acid-reactive material in a dose-dependent manner. Similar results were obtained in an aqueous system containing phospholipid vesicles initiated by Fe/ascorbate. For both systems I-3-C was less effective than tocopherol or BHT as antioxidant. To assess these antioxidant effects in vivo, mice were treated with I-3-C by gavage. A hepatic post-mitochondrial supernatant fraction isolated 2 hours after treatment showed dose-dependent decreases in NADPH-mediated lipid oxidation which correlated with decreases in 14C-nitrosodimethylamine covalent binding to protein. Although hepatotoxicity may not involve lipid oxidation per se, it does indicate that free radical damage had occurred. Inhibition of damage by I-3-C suggests that this dietary component has the potential to ameliorate radical mediated chemical toxicity.

ISOLATION OF MUTAGENIC COMPOUNDS FROM SLUDGES AND WASTEWATERS

Extracts of sludges and effluent wastewaters from municipal sewage treatment plants have been shown to be complex mixtures of organic chemicals (e.g., Baird et al., 1980; Bedding et al., 1982; Ellis et al., 1982; Johnston et al., 1982; Naylor and Loehr, 1982a, b; Clevenger et al., 1983; Hrubec et al., 1983; Strachan et al., 1983). During the past few years, these residue organics have been characterized as toxic and/or mutagenic in a variety of bacterial, animal, and plant test systems (e.g., Rappaport et al., 1979; Baird et al., 1980; Neal et al., 1980; Babish et al., 1982; Hopke et al., 1982; Johnston et al., 1982; Babish et al., 1983; Clevenger et al., 1983; Hopke and Plewa, 1983; Hrubec et al., 1983; Maciorowski et al., 1983; Hopke et al., in press; Meier and Bishop, 1984). The major mutagens appeared to be in the nonvolatile residue organic materials from sewages that included industrial sources. Although many volatile compounds, including priority pollutants, have been identified and quantified (e.g.. Bedding et al., 1982; Harrold et al., 1982; Johnston et al., 1982; Naylor and Loehr, 1982a, b; Petrasek et al., 1983; Strachan et al., 1983), the chemical identity and source of the vast majority of the mutagens is unknown (Nellor et al., 1982).

Effects of metyrapone on microsomal-dependent Salmonella mutagenesis: Studies with chloroallyl ethers and model compounds

Metyrapone (2-methyl-1,2-di-3-pyridyl-1-propanone, MTP) is used as an inhibitor of cytochrome P-450 enzymes, particularly those induced by phenobarbital (PB). We examined the effects of MTP on the microsomal dependent mutagenesis of a newly isolated promutagen, 3-(2-chloroethoxy)-1,2-dichloropropene (CP), three S-chloroallyl thiocarbamate herbicides, and four model promutagens aflatoxin B1 (AFB), 2-acetylaminofluorene (2AAF), 2-aminoanthracene (2AA) and benzo[a]pyrene (BP). Salmonella tester strains TA98, TA100 and TA1535 and liver microsomal preparations (S9) from rats induced with PB or Aroclor 1254 (PCB) were employed. For statistical analysis, mutagenesis data were transformed and subjected to two-way analysis of variance. Metyrapone alone was not mutagenic in the absence or presence of S9. In a dose-dependent manner, MTP inhibited mutagenesis of AFB for strains TA98 and TA100 and enhanced mutagenesis of 2AAF, 2AA and BP for these strains. 3-(2-Chloroethoxy)-1, 2-dichloropropene and the herbicides diallate, triallate and sulfallate are all chloroallyl ethers. They are similar in their mutagenesis for Salmonella with respect to strain specificity, mutagenic potency, and requirement for activation by specifically-induced microsomes. Metyrapone inhibited the mutagenesis of CP, triallate and sulfallate for strain TA100 in the presence of either PB- or PCB-induced S9, and had no apparent effect on diallate mutagenesis; the same results were obtained for TA1535 with PCB-induced S9. On this basis, the mutagenic activation of diallate appears to be different from that of the other chloroallyl ethers tested. Our results indicate that MTP can inhibit as well as enhance microsomal dependent mutagenesis for Salmonella. We conclude that MTP may be a useful tool in characterizing pathways for promutagen activation.

Chemical Analysis for Assessment and Evaluation of Environmental Pollutants: Fact or Artifact

Due to the presence of both known and unknown sites and sources of environmental pollutants in many industrialized regions, there exists the possibility for releases of hazardous anthropogenic organics into the environment that present a potential hazard to human health. Many of these releases remain undetected until after the fact; i.e., endpoints of exposure surface in the indigenous population, chemical monitoring/biological monitoring or survey programs show a contamination or threat of release, or the pollutants affect the flora of fauna at the site of the contamination or release. An approach to early detection of environmental pollutants or release of these pollutants is the use of in situ monitoring methodologies via sentinel organisms. These situations require an approach by the environmental analytical chemist that provides data appropriate to assess and evaluate the situation so that effective measures can be instituted. The acquisition of such data requires analyses at trace levels, i.e., ppb or lower, thereby increasing the probability of interferences from artifacts of isolation or inadequately resolved components in the complex mixture under analysis. Considering these problems, an overall analytical strategy and an understanding of the analytical process is required for such cases of assessment and evaluation of environmental pollutants.