I.G. Sipes

In vitro solubility and in vivo toxicity of gallium arsenide

The in vitro solubilities of gallium arsenide (GaAs) and its metal oxides were arsenic(III) oxide greater than GaAs much greater than gallium(III) oxide. GaAs dissolution was also dependent upon the type and concentration of buffer anion. The amount of arsenic dissolved in 12 hr by various aqueous media was 0.2 M phosphate buffer greater than or equal to 0.1 M phosphate buffer greater than Krebs-Hensleit buffer greater than distilled H2O greater than HCl-KCl buffer. GaAs was apparently soluble under in vivo conditions. Blood arsenic concentrations in rats 14 days after intratracheal instillation of 10, 30, or 100 mg/kg GaAs were 5.5, 14.3, and 53.6 micrograms/ml, respectively; gallium was not detected at any doses. An increase in lung wet weight at 14 days was dose dependent with these organs retaining 17 to 42% of the dose as gallium or arsenic. Excretion of gallium and arsenic was limited to the feces. Urinary porphyrin concentrations and body weight, monitored as indices of toxicity, were significantly altered over the 14-day study. The analysis of porphyrins revealed that uroporphyrin replaced coproporphyrin as the primary urinary metabolite. Rats receiving 10, 100, or 1000 mg/kg GaAs po exhibited similar signs of toxicity. Blood arsenic concentrations at 14 days were 3.5, 6.8, and 17.6 micrograms/ml, respectively. Porphyria was increased, and body weight was decreased at 1000 mg/kg GaAs. These values were equivalent to those obtained with an intratracheal dose of 10 to 30 mg/kg GaAs. Our results showed that pulmonary and po exposure to GaAs resulted in systemic arsenic intoxication. The finding that urinary uroporphyrin concentrations were greater than coproporphyrin concentrations may serve as a sensitive indicator for GaAs exposure.

The acute hepatotoxicity of the isomers of dichlorobenzene in Fischer-344 and Sprague-Dawley rats: Isomer-specific and strain-specific differential toxicity

The acute hepatotoxicity of the three isomers of dichlorobenzene (DCB) was evaluated in male Fischer-344 (F344) rats at various times following ip administration. Plasma alanine aminotransferase (ALT) activity, measured in F344 rats 24 hr postexposure, was dramatically elevated following doses of 1.8-5.4 mmol/kg of o-DCB. Conversely, equimolar doses of p-DCB produced no such toxicity, while m-DCB produced intermediate hepatic injury at or above doses of 2.7 mmol/kg. Histopathological changes in livers from treated animals qualitatively reflected elevations in 24-hr plasma ALT activity (time to maximal elevation). Phenobarbital pretreatment potentiated the acute hepatotoxicity of o- and m-DCB, but did not affect the toxicity of p-DCB. Likewise, SKF-525A pretreatment inhibited the hepatotoxicity of o-DCB. Equimolar doses of o- and m-DCB produced approximately equivalent depletion of intrahepatic glutathione, while p-DCB had no effect on hepatic GSH. Furthermore, prior depletion of hepatic glutathione by pretreatment with phorone markedly potentiated the hepatotoxicity of o- and m-DCB, while increasing the toxicity of p-DCB to a far lesser degree. The differential hepatotoxicity of the o- and m-DCB does not appear to be explained adequately by differences in their hepatic distribution or in vivo covalent binding to hepatic proteins. Interestingly, male Sprague-Dawley (SD) rats are relatively refractive to the acute hepatotoxicity of o-DCB following ip administration of 1.8 and 5.4 mmol/kg. The combination of these dramatic differences (structure-activity and animal strains) should be useful in elucidating key events involved in the hepatotoxicity caused by these compounds.

Gadolinium chloride reduces cytochrome P450: relevance to chemical-induced hepatotoxicity.

The Kupffer cell inhibitor, gadolinium chloride (GdCl3), protects the liver from a number of toxicants that require biotransformation to elicit toxicity (i.e. 1,2-dichlorobenzene and CCl4), as well as compounds that do not (i.e. cadmium chloride and beryllium sulfate). The mechanism of this protection is thought to result from reduced secretion of inflammatory and cytotoxic products from Kupffer cells (KC). However, since other lanthanides have been shown to decrease cytochrome P450 (P450) activity, the following studies were designed to determine if GdCl3 pretreatment alters hepatic P450 levels or activity. The toxicological relevance of GdCl3-mediated alterations in P450 activity was also estimated by determining the effect of GdCl3 pretreatment on the susceptibility of primary cultured hepatocytes to CCl4 and cadmium chloride (CdCl2). Male and female Sprague-Dawley rats were given GdCl3 (i.v., 10 mg/kg). Twenty-four hours later, livers were either processed for preparation of microsomes or for primary cultures of hepatocytes. Gadolinium chloride treatment reduced total hepatic microsomal P450 as well as aniline hydroxylase activity by approximately 30% in males and 20% in females. In hepatocytes isolated from rats pretreated with GdCl3, the toxicity caused by CCl4, but not CdCl2 was reduced. Interestingly, when GdCl3 was administered in vitro to microsomes, there was no effect on either the microsomal P450 difference spectra or p-hydroxylation of aniline. However, when GdCl3 was incubated with isolated hepatocytes, the cytotoxicity of CCl4 (but not CdCl2) was partially attenuated. These results suggest that, in addition to its inhibitory effects on KC, GdCl3 produces other effects which may alter the susceptibility of hepatocytes to toxicity caused by certain chemicals.

Microsomal bioactivation and covalent binding of aliphatic halides to DNA.

Studies were carried out on the in vitro covalent binding of a series of 14C-labeled aliphatic halides to calf thymus DNA following bioactivation by hepatic microsomes isolated from phenobarbital-treated rats. Six compounds were shown to exhibit binding to DNA of greater than 0.3 nmol/mg DNA (1,2-dibromoethane, bromotrichloromethane, trichloroethylene, carbon tetrachloride, chloroform, and 1,1,2-trichloroethane). Covalent binding of the aliphatic halides to the nucleic acids was confirmed by sedimentation of the DNA-organohalogen adduct in a cesium chloride gradient and Sephadex LH-20 chromatography of the nucleosides released by enzymatic hydrolysis.

Comparison of the disposition and in vitro metabolism of 4-vinylcyclohexene in the female mouse and rat

4-Vinylcyclohexene (VCH) is a chemical to which humans are exposed in the rubber industry. A chronic carcinogenicity bioassay conducted by the National Toxicology Program showed that oral administration of VCH induced tumors in the ovaries of mice but not in those of rats. The hypothesis tested was that the species and organ specificity of VCH toxicity was due to differences in the disposition of VCH between the female rat and mouse. Therefore, the disposition of a single oral dose of 400 mg/kg [14C]VCH was studied in female B6C3F1 mice and Fischer 344 rats. Mice eliminated greater than 95% of the dose in 24 hr, whereas rats required 48 hr to eliminate greater than 95% of the dose. The major routes of excretion of [14C]VCH-derived radioactivity were in the urine (50-60%) and expired air (30-40%). No evidence was obtained to indicate that the ovaries of either species retained VCH as a parent compound or as radioactive equivalents. A dramatic difference was observed between the rat and mouse in the appearance of a monoepoxide of VCH in blood from 0.5 to 6 hr after VCH administration (800 mg/kg, ip). VCH-1,2-epoxide was present in the blood of mice with the highest concentration at 2 hr (41 nmol/ml). The blood concentration of VCH-1,2-epoxide in rats was less than 2.5 nmol/ml at all times examined. VCH-7,8-epoxide was not present in the blood of either species at the level of detection. These findings were supported by in vitro studies of VCH epoxidation by liver microsomes. The rate of epoxidation of VCH (1 mM) to VCH-1,2-epoxide was 6.5-fold greater in mouse liver microsomes than that in rat liver microsomes. The species difference in the rate of epoxide formation by the liver may be an important factor in the species difference in susceptibility to VCH-induced ovarian tumors.

Deuterium isotope effect on the metabolism and toxicity of 1,2-dibromoethane

The metabolism, hepatotoxicity, and hepatic DNA damage of 1,2-dibromoethane (EDB) and tetradeutero-1,2-dibromoethane (d4EDB) were compared in male Swiss-Webster mice. In vitro studies that measured bromide ion released from the substrate to monitor the rate of metabolism showed that the hepatic microsomal metabolism of EDB was significantly reduced by deuterium substitution, while metabolism by the hepatic glutathione S-transferases was unaffected. Three hours after ip administration of EDB or d4EDB (50 mg/kg), there was 42% less bromide in the plasmaa of d4EDB-treated mice than in the plasm of EDB-treated mice. This difference demonstrates a significant deuterium isotope effect on the metabolism of EDB in vivo. Although the metabolism of d4EDB was less than that of EDB 3 hr after exposure, the DNA damage caused by both analogs was not significantly different at this time point. At later time points (8, 24, and 72 hr), d4EDB caused significantly greater DNA damage than EDB. Since the decreased metabolism of d4EDB was apparently due to a reduced rate of microsomal oxidation, these data support the hypothesis that conjugation with GSH is responsible for the genotoxic effects of EDB.

Comparative metabolism and toxicity of dichlorobenzenes in Sprague-Dawley, Fischer-344 and human liver slices

1 Precision-cut liver slices, prepared from Sprague- Dawley and Fischer-344 rats and donated human liver tis sue, were used to identify differences in 1,2-dichloroben zene (1,2-DCB), 1,3-dichlorobenzene (1,3-DCB) and 1,4- dichlorobenzene (1,4-DCB) metabolism and how it may relate to toxicity. 2 Rat and human liver slices were incubated with 1 mM of either dichlorobenzene to determine metabolism and toxi city, at 2 and 6 h of organ culture. 3 The human liver slices metabolised the dichloroben zenes to a greater extent than those from either of the rat strains. Liver slices from the Fischer-344 strain had a higher metabolic rate than the slices from the Sprague- Dawley rat strain. 4 The metabolic rate of dichlorobenzene isomers did not consistently correlate with its toxicity. For example, human slices did not exhibit any hepatotoxicity, even though they metabolised these compounds to a greater extent than either rat strain. 5 Cross species covalent binding did not correlate with toxicity endpoints measured in this study. 6 The phase two metabolite profiles for each of the iso mers in human and rat slices were similar in that the glu tathione-cysteine conjugate was the major metabolite. 7 The use of an in vitro system which utilises human liver slices might provide an important bridge between animal derived data and the human situation.

Factors affecting the formation of chlorotrifluoroethane and chlorodifluoroethylene from halothane

Since CF3CH2Cl and CF2CHCl are probably the products of reactive intermediates formed during the reductive metabolism of halothane (CF3CHClBr), factors affecting their in vitro and in vivo formation were investigated. In vitro studies with rat hepatic microsomes showed that CF3CH2Cl and CF2CHCl are produced by cytochrome P-450 mediated reductive pathways which were inhibited by the presence of CO. Under conditions of exposure known to promote halothane hepatotoxicity in phenobarbital treated rats (1 per cent halothane, 14 per cent oxygen), the hepatic and blood concentrations of the volatile metabolites were enhanced. Central venous levels of the volatile metabolites were much higher than the concentration in peripheral vessels. The CF3CH2Cl/CF2CHCl ratio in blood was approximately three, whereas the ratio in vitro was almost unity. Liver levels of the two volatile metabolites greatly exceeded the blood levels, but interestingly they were present in equivalent concentrations. The differences in the ratio of CF3CH2Cl to CF2CHCl may be explained by the fact that CF2CHCl is further degraded under oxidative conditions, whereas CF3CH2Cl appears relatively stable. Measurement of these metabolic products in patients undergoing halothane anesthesia may permit rapid detection of an unusually high level of halothane biotransformation along its hepatotoxic pathway.

Absorption, distribution, metabolism and excretion of intravenously and orally administered tetrabromobisphenol A [2,3-dibromopropyl ether] in male Fischer-344 rats.

Tetrabromobisphenol A bis[2,3-dibromopropyl ether],2,2-bis[3,5-dibromo-4-(2,3-dibromopropoxy)phenyl]propane is a brominated flame retardant with substantial U.S. production. Due to the likelihood of human exposure to TBBPA-DBPE and its probable metabolites, studies regarding the absorption, distribution, metabolism, and excretion were conducted. Male Fischer-344 rats were dosed with TBBPA-DBPE (20mg/kg) by oral gavage or IV administration. Following a single oral administration of TBBPA-DBPE, elimination of [(14)C] equivalents in the feces was extensive and rapid (95% of dose by 36h). Following repeated daily oral doses for 5 or 10 days, route and rate of elimination was similar to single administrations of TBBPA-DBPE. After IV administration, fecal excretion of [(14)C] equivalents was much slower (27% of dose eliminated by 36h, 71% by 96h). Urinary elimination was minimal (<0.1%) following oral or IV administration. A single peak that co-eluted with the standard of TBBPA-DBPE was detected in extracts of whole blood following oral or IV administration. TBBPA-DBPE elimination from the blood was slow. Kinetic constants following IV dosing were-t(1/2beta): 24.8h; CL(b): 0.1mLmin(-1). Kinetic constants following oral dosing were: t(1/2alpha): 2.5h; t(1/2beta): 13.9h; CL(b): 4.6mLmin(-1). Systemic bioavailability was 2.2%. Liver was the major site of disposition following oral or IV administration. After oral administration, 1% of the dose was eliminated in bile in 24h (as metabolites). In in vitro experiments utilizing hepatocytes or liver microsomal protein, no detectable metabolism of TBBPA-DBPE occurred. These data indicate that TBBPA-DBPE is poorly absorbed from the gastrointestinal tract. Compound which is absorbed is sequestered in the liver, slowly metabolized, and eliminated in the feces.

Comparison of the requirements for hepatic injury with halothane and enflurane in rats.

A rat model of cnflurane-associated hepatotoxicity was compared with the halothane-hypoxia (HH) model (adult male rats, phenobarbital induction, 1% halothane, 14% O2, for 2 hr). The enflurane-hypoxia heating (EHH) model involved exposing phenobarbital-pretreated male adult rats to 1.5–1.8% enflurane at 10% O2 for 2 hr with external heating to help maintain body temperature. Exposure to either anesthetic without temperature support led to a decrease in body temperature of 7–9°C, while heating the animals during anesthesia resulted in only a 0.5–2°C decrease. Reducing the oxygen tension to 10% O2 combined with heating the animals during exposure produced significant decreases in the oxidative metabolism of both halothane and enflurane as compared to exposures of 14% O2. The same conditions also caused a significant increase in the reductive metabolism of halothane, indicating that a severe hepatic hypoxia or anoxia occurs during anesthesia at 10% O2 with external heating. The time course of lesion development in the HH model paralleled results obtained with an oral dose of CCl4: gradual progression of necrosis up to 24 hr. EHH resulted in a classic hypoxic/anoxic injury with elevated serum glutamate pyruvate transaminase values and a watery vacuolization of centrilobular hepatocytes immediately after exposure. The HH model required phenobarbital pretreatment of the rats for expression of hepatic injury; EHH did not. Heating of the animals during anesthesia exposure was necessary for enflurane-induced hepatotoxicity but had little effect on the HH model. Exposure to 5% O2 without anesthetic mimicked EHH in both requirements for and type of hepatic injury. Thus the HH model appears to act via a bioactiva- tion/chemotoxic mechanism, whereas models of anesthetic- induced hepatotoxicity that require very low oxygen tensions and heating of the animals during exposure result from a severe hypoxia/anoxia of the liver.