Disposition and metabolism of isoeugenol in the male Fischer 344 rat.

The primary objective of these studies was to determine the absorption, distribution, metabolism and excretion of isoeugenol following oral and intravenous administration to male Fischer-344 rats. Following a single oral dose of [14C]isoeugenol (156 mg/kg, 50 microCi/kg), greater than 85% of the administered dose was excreted in the urine predominantly as sulfate or glucuronide metabolites by 72 h. Approximately 10% was recovered in the feces, and less than 0.1% was recovered as CO(2) or expired organics. No parent isoeugenol was detected in the blood at any of the time points analyzed. Following iv administration (15.6 mg/kg, 100 microCi/kg), isoeugenol disappeared rapidly from the blood. The t(1/2) was 12 min and the Cl(s) was 1.9 l/min/kg. Excretion characteristics were similar to those of oral administration. The total amount of radioactivity remaining in selected tissues by 72 h was less than 0.25% of the dose following either oral or intravenous administration. Results of these studies show that isoeugenol is rapidly metabolized and is excreted predominantly in the urine as phase II conjugates of the parent compound.

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.

The role of inflammatory cells and cytochrome P450 in the potentiation of CCl4-induced liver injury by a single dose of retinol.

Evidence suggests that 7 days of retinol pretreatment potentiates chemical-induced liver injury by a mechanism that involves activation of Kupffer cells (KC). These studies were designed to determine if shorter dosing regimens of retinol potentiate carbon tetrachloride (CCl4). Initially, a single dose of retinol was shown to potentiate the hepatotoxicity of CCl4. Male Sprague-Dawley rats were pretreated with all-trans-retinol (75 mg/kg p.o.) 24 hr prior to KC isolation or administration of CCl4 (0.2 ml/kg i.p.). KC isolated at 24 hr after retinol released increased amounts of superoxide anion when stimulated with zymosan or phorbol myristate acetate. At 24 hr after CCl4, plasma ALT activities and histological sections of liver were examined. Retinol-pretreated rats showed a significant elevation in both enzyme leakage and centrilobular to midzonal necrosis compared to retinol vehicle controls following CCl4. Although complete protection was not seen, depletion of KC or neutrophils (PMNs) (by gadolinium chloride (GdCl3) or a PMN-depleting antibody, respectively) significantly reduced the hepatotoxicity of 1 day retinol/CCl4 liver injury. Immunohistochemical analysis of livers showed significant elevations in positive staining for ED2, ED1, and HIS48 in retinol-pretreated rats given CCl4. GdCl3 effectively reduced ED2 staining but did not greatly affect HIS48 staining. Additional studies were performed to estimate the effect of retinol on noninflammatory processes. While total cytochrome P450 was not increased, the activity and concentration of CYP2E1 were both significantly elevated after a single dose of retinol. Hepatocytes isolated from 1-day retinol-treated rats were also more susceptible to CCl4 injury, a consequence that is most likely related to elevated CYP2E1 activity. These findings suggest that a single pretreatment with retinol may potentiate CCl4 hepatotoxicity by multiple mechanisms which involve increased biotransformation and inflammatory cell activities.

Vitamin A potentiation of vinylidene chloride hepatotoxicity in rats and precision-cut rat liver slices.

Pretreatment of large doses of vitamin A (VA) is known to potentiate the hepatotoxicity of carbon tetrachloride. Therefore the effects of 1-day VA pretreatment on VDC hepatotoxicity was examined both in vivo and in an in vitro system of precision-cut rat liver slices. Male Sprague-Dawley rats were pretreated with 250,000 IU/kg VA by oral gavage. After 24 hr rats were administered 50, 100, or 200 mg/kg VDC ip. Precision-cut liver slices were prepared from VA pretreated rats 24 hr later and the liver slices were exposed for 2-8 hr to 0.025-1.0 microliter VDC evaporated into the gas phase of the incubation vials. VA pretreatment resulted in an enhancement of VDC toxicity, both in vivo and in vitro. There was a dose-dependent increase in plasma ALT 24 hr after VDC treatment of rats and an increase in K+ leakage from liver slices after VDC exposure. Histological analysis of the liver or the liver slices revealed that VA + VDC treatment resulted in centrilobular necrosis of the liver. When GdCl3 (10 mg/kg iv) was administered just before VA pretreatment of rats, VDC toxicity was partially reversed as observed by a decrease in ALT in vivo and a decrease in the loss of K+ in vitro. These results indicated that Kupffer cells, the resident macrophages of the liver, were partially responsible for the VA-potentiated VDC hepatotoxicity. One-day pretreatment of VA induced cytochrome P450IIE1 protein content as well as its enzymatic activity as measured by pnitrophenol hydroxylation. Because VDC is bioactivated by cytochrome P450IIE1, the increase in VDC hepatotoxicity after VA may be due to an increased bioactivation of VDC in the liver and in precision-cut liver slices. Thus, more than one mechanism may be involved in the VA enhancement of VDC hepatotoxicity.

Alterations in chemically induced tissue injury related to all-trans-retinol pretreatment in rodents.

Retinol (vitamin A) is an essential nutrient which has many physiological effects throughout the body. Our studies have demonstrated that retinol modulation of immune response, through alteration of macrophage and neutrophil function, can have dramatic effects on the toxicity of some compounds. Based on these studies, our current hypothesis for retinol potentiation of chemical-induced liver injury is that retinol administered to rats prior to the hepatotoxicant (CCl4 and AA in rats; and AA, APAP, and GalN in mice) primes the Kupffer cells to a more active state. This may occur in part as a result of increases in chemical mediators such as TNF from these Kupffer cells. Following hepatocyte damage by a toxicant, Kupffer cells are activated to release reactive oxygen species, immune mediators, and chemotactic factors which all serve to enhance the inflammatory response. This increased inflammatory response then results in increased injury to the already toxicant-damaged hepatocytes. In addition, retinol modulation of toxicant activation and detoxification may also make important contributions to the potentiation of some toxicants such as AA. Retinol protection of CCl4 hepatotoxicity in mice is more difficult to explain at this time but is possibly related to alterations in CCl4 metabolism in this species. Differences in response between pulmonary and liver macrophages (Kupffer cells) may explain the retinol protection from 1-NN pulmonary toxicity. Retinol may decrease the inflammatory response through downregulation of pulmonary macrophage function, thus resulting in decreased pulmonary injury. Finally, since retinol protection of cadmium toxicity in the liver and testis requires 7 days of retinol pretreatment, we suspect that retinol is inducing protective protein(s) in these organs. Aside from its normal biological role in rhe body, clinical medicine has found new uses for retinol in the treatment and prevention of some cancers, and in the treatment of certain dermatologic conditions. Since these patients are frequently administered or exposed to other potentially toxic compounds, it is obviously prudent and necessary to continue research into the effects of retinol on immune modulation and interaction with other compounds. More importantly, these studies demonstrate the modulation of immune function is one mechanism by which one chemical can influence the toxicity of another.