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Biomedical subjects

I G Sipes

Publications and source records attributed to I G Sipes.

At least 37 records · Page 2Linked to original sources
PubMed1999

A single dose of the ovotoxicant 4-vinylcyclohexene diepoxide is protective in rat primary ovarian follicles.

Repeated dosing of rats with the ovotoxic chemical, 4-vinylcyclohexene diepoxide (VCD), destroys primordial and primary ovarian follicles via apoptosis (physiological cell death) by accelerating the normal rate of atresia. The present study investigated the effect of a single dose (1x) of VCD. Immature (d28) female Fischer 344 rats were dosed 1x or 15x with VCD (80 mg/kg ip). Ovaries were collected 24 h or 15 days following 1x VCD or after 15x for classification and evaluation. Following 1x VCD the number of healthy primary follicles was greater (p < 0.05) than control 24 h and 15 days later. This effect reflected a slowing of the normal rate of atresia seen in control ovaries. There was no effect of a single dose on primordial or growing follicles at any time. Expression of mRNA encoding the cell death gene bax was reduced (p < 0.05) on d1 after 1x VCD in isolated primordial and primary follicles. These observations were in contrast to a decreased (p < 0. 05) number of healthy primary and primordial follicles in ovaries and increased (p < 0.05) bax mRNA in isolated follicles from rats dosed 15x for 15 days. Immunofluorescence staining revealed that, the distribution of Bax protein was similar between ovaries from controls and 1x or 15x VCD-treated rats. These data provide evidence for a "protective" response against the normal rate of atresia in primary ovarian follicles following exposure to 1x VCD. Additionally, changes in expression of bax mRNA paralleled alterations in the rate of atresia.

Animals↗
PubMed1999

Role of cytochrome P4502E1 in retinol's attenuation of carbon tetrachloride-induced hepatotoxicity in the Swiss Webster mouse.

In the mouse, retinol administration attenuates carbon tetrachloride (CCl4)-induced hepatic injury. We have investigated the role of cytochrome P4502E1 (CYP2E1) in this interaction. Male Swiss Webster mice were administered retinol (75 mg/kg/d) or vehicle for 3 days prior to CCl4 (30 microl/kg, ip). Hepatotoxicity produced by CCl4 was assessed by plasma alanine aminotransferase (ALT) activity and light microscopy (ALT activity of 1391+/-430 vs. 274+/-92 IU/L for vehicle + CCl4 and retinol + CCl4 treatments respectively, p < 0.05). Retinol's attenuation of liver injury was maintained when CCl4 was administered 48 h after the conclusion of the retinol pretreatment. Aniline hydroxylation activity, an indicator of CYP2E1 catalytic activity, determined on day 4 was 33.8% of untreated control in vehicle + CCl4 treatments while the retinol + CCl4 treatment group was 94.2% of untreated control. Additionally, CYP2E1 immunoreactive protein was 78% lower in vehicle + CCl4 vs. retinol + CCl4 treatment groups. Attenuation of potentiated hepatotoxicity was also observed when CYP2E1 was induced by acetone (ALT activity of 3119+/-1066 vs. 247+/-77 IU/L for vehicle and retinol treatments respectively, p < 0.05). In the mouse, retinol itself does not alter constitutive or inducible CYP2E1 expression. However, in combination with CCl4 retinol does reduce the amount of CCl4 bioactivated to its toxic metabolite. We conclude that retinol attenuates CCl4-induced hepatotoxicity by causing a decrease in CCl4 bioactivation but does not cause a decrease in CYP2E1 expression.

Animals↗
PubMed1999

Metabolism and disposition of [(14)C]1-nitronaphthalene in male Sprague-Dawley rats.

In rats and mice, 1-nitronaphthalene (1-NN) produces both lung and liver toxicity. Even though these toxicities have been reported, the metabolism and disposition of 1-NN have not been elucidated. Therefore, studies were performed to characterize its fate after i.p. and i.v. administration to male Sprague-Dawley rats. After i.p. administration of [(14)C]1-NN (100 mg/kg; 60 microCi/kg), 84% of the dose was eliminated in the urine and feces by 48 h. At 96 h, 60% of the dose was recovered in the urine, 32% in the feces, and 1% collectively in the tissues, blood, and gastrointestinal contents. The terminal phase rate constant (k(term)) of 1-NN was 0.21 h(-1), the terminal phase half-life (T(1/2,term)) was 3.40 h, and the systemic bioavailability was 0.67. When administered i.v. (10 mg/kg; 120 microCi/kg), 85% of the dose was eliminated in the urine and feces by 24 h. At the end of the study (96 h), 56% of the dose was recovered in the urine, 36% in the feces, and 1% collectively in the tissues, blood, and gastrointestinal contents. Interestingly, 88% of the dose was secreted into bile by 8 h. The k(term) was 0.94 h(-1) and the T(1/2,term) was 0.77 h. The major urinary metabolite after both routes of administration was N-acetyl-S-(hydroxy-1-nitro-dihydronaphthalene)-L-cysteine. Other urinary metabolites identified include hydroxylated, dihydroxylated, glucuronidated, sulfated, and reduced metabolites, as well as dihydrodiol. The major biliary metabolite was hydroxy-glutathionyl-1-nitro-dihydronaphthalene. These data show that 1-NN undergoes extensive metabolism and enterohepatic recirculation, and the majority of the dose is eliminated in the urine.

Animals↗
PubMed1999

Induction of cytochrome P-450 enzymes after repeated exposure to 4-vinylcyclohexene in B6C3F1 mice.

4-Vinylcyclohexene (VCH), an ovarian toxicant in mice, is known to irreversibly deplete ovarian follicles as a consequence of VCH diepoxide formation. Because ovotoxicity requires repeated dosing of VCH, the effect of consecutive daily doses of VCH (7.5 mmol/kg/day) on mouse liver microsomal activities and VCH epoxidation was determined. Cytochromes P-450 2B and 2A (CYP2B and CYP2A), principle isoforms involved in the bioactivation of VCH, as well as CYP2E1 and CYP3A were evaluated. VCH exposure increased total cytochrome P-450 content (35-83% above control levels) after either 5, 10, or 15 days of treatment. Western blot analysis revealed an induction of CYP2A, CYP2B, and CYP2E1 at day 10. Elevated levels of CYP2A and CYP2B correlated with marker androstenedione and testosterone 16alpha- and 16beta-hydroxylase activities. Microsomes prepared from mice pretreated with VCH for 10 days demonstrated an increase (>/=2-fold) in the rate of VCH monoepoxide and diepoxide formation. Microsomal VCH epoxidation was increased to a similar extent by phenobarbital, acetone, and dexamethasone treatment. An increase in cytosolic glutathione S-transferase activity was observed after repeated VCH treatment, an enzyme potentially involved in detoxification of the VCH epoxides. Interestingly, preliminary studies indicated that circulating levels of the monoepoxide (vinylcyclohexene 1, 2-monoepoxide) and diepoxide of VCH were elevated after repeated dosing of VCH. Overall, the results indicate that repeated exposure of VCH in mice induces cytochrome P-450-dependent activities, and in turn induction of its metabolism. Additional studies examining the toxicokinetics of VCH after repeated exposure are required to further delineate the relevance of induction in VCH-induced ovotoxicity.

Animals↗
PubMed1998

Alteration of Kupffer cell function and morphology by low melt point paraffin wax in female Fischer-344 but not Sprague-Dawley rats.

This study was conducted to compare the effects of 60-day dietary exposure (2%) to low melt point paraffin wax (LMPW) on both general liver morphology and Kupffer cell (KC) function and morphology in female F-344 and Sprague-Dawley (SD) rats. Livers from only F-344 rats fed LMPW had granuloma formation/lymphoid cell aggregates with small areas of necrosis. Significant increases in serum alanine and aspartate aminotransferase as well as gamma-glutamyltransferase activities were detected only in treated F-344 rats. Additionally, detectable amounts of LMPW were present only in livers of treated F-344 rats. Because KC can be involved in granuloma formation, their morphology and function were examined. Electron microscopy revealed the presence of large, irregularly shaped, membrane-associated vacuoles in cells isolated from F-344 rats exposed to LMPW. These vacuoles were not seen in KC from control rats and rarely detected in KC isolated from LMPW-exposed SD rats. Moreover, indices of KC function including phagocytic activity and nitric oxide and superoxide anion production were significantly increased by KC isolated from F-344 rats exposed to LMPW (1.6-, 36-, and 2.2-fold increases, respectively) over untreated controls. In contrast, LPS-stimulated production of TNF and LTB4 was significantly decreased only in KC of LMPW-fed F-344 rats. No significant changes in these functions were observed in KC isolated from SD rats exposed to LMPW or from KC isolated from control F-344 or SD rats. These data provide evidence that dietary LMPW alters the morphology and functional capacity of KC of F-344 but not SD rats and these changes may ultimately lead to granuloma formation.

Animals↗
PubMed1998

1,2-Dichlorobenzene-induced lipid peroxidation in male Fischer 344 rats is Kupffer cell dependent.

1,2-Dichlorobenzene (1,2-DCB) is a potent hepatotoxicant in male Fischer 344 (F344) rats and previous studies have suggested that reactive oxygen species may play a role in the development of hepatotoxicity. Since reactive oxygen species can damage lipid membranes, this study was conducted to determine the extent of lipid peroxidation after administration of 1,2-DCB by immuno-histochemical analysis of 4-hydroxynonenal (4-HNE) protein adduct formation in liver and conjugated diene formation in liver and serum. The contribution of Kupffer cells to the lipid peroxidation was also investigated. Male F344 rats were administered 1,2-DCB (3.6 mmol/kg i.p. in corn oil) and killed at selected times between 3 and 48 h. Time course studies revealed the greatest abundance of 4-HNE protein adducts in the centrilobular regions of the liver 24 h after 1,2-DCB administration, with much lower levels at 16 h. Adducts were present in necrotic and vacuolized centrilobular hepatocytes of 1,2-DCB treated rats but not in livers of controls. Further, conjugated dienes were significantly increased in liver and serum 16 and 24 h after 1,2-DCB administration, peaking at 24 h. These data correlated with hepatocellular injury, determined by serum alanine aminotransferase activity and histopathological evaluation, which was markedly elevated within 16 h and peaked at 24 h. When rats were pretreated with gadolinium chloride (GdCl3; 10 mg/kg i.v. 24 h prior to 1,2-DCB), an inhibitor of Kupffer cells, hepatotoxicity was decreased by 89 and 86%, at 16 and 24 h, respectively. Conjugated diene concentrations were decreased to control values at these times after 1,2-DCB administration. Moreover, no 4-HNE protein adducts were detected in livers of 1,2-DCB-treated rats pretreated with GdCl3. Finally, Kupffer cells isolated from 1,2-DCB-treated rats produced significantly more superoxide anion than Kupffer cells isolated from vehicle controls. These data, along with previous findings, suggest that lipid peroxidation associated with 1,2-DCB is mediated in part by Kupffer cell-derived reactive oxygen species.

Alanine Transaminase↗
PubMed1998

Reduction of thyroid hormone may participate in the modulation of cytochromes P450 2C11 and 3A2 by retinol.

Both hypothyroidism and retinol supplementation in rats induce CYP 3A2 and suppress CYP 2C11. Therefore studies were performed to evaluate the role of thyroid hormones in the modulation of P450 expression by retinol. Adult male Sprague-Dawley rats were given retinol as a single oral dose of 75 mg/kg. Rats were killed and hepatic microsomes prepared at 24, 48, 72, and 96 hr following treatment. The catalytic activity of 2C11 was reduced maximally by retinol at 48 hr (by 30%) whereas 3A2 activity was elevated maximally at 24 hr (by 30%). The serum concentration of testosterone was not altered at any time point. However, retinol produced a decline in the concentration of thyroxine by 35% and 43% at 24 and 48 hr, respectively. These data suggest that administration of large doses of retinol may alter hepatic microsomal enzyme expression by perturbation of plasma thyroid hormone levels.

Animals↗
PubMed1998

Metabolic and dispositional fate of 1,2-dibromo-2,4-dicyanobutane in the male fischer 344 Rat.

Studies were conducted to characterize the absorption, disposition, metabolism, and excretion of 1,2-dibromo-2,4-dicyanobutane (BCB; methyldibromoglutaronitrile) following iv, oral, and topical administration to male Fischer 344 rats. Following iv administration of [14C]BCB (8 mg/kg, 120 microCi/kg), no parent compound was detected in the blood; however, its debrominated metabolite, 2-methyleneglutaronitrile (2-MGN; Cmax 7.3 micrograms/ml), was observed up to 1 hr. Within 72 hr, greater than 60% of the dose was excreted in the urine and 4.1% in the feces, and 6.6% was exhaled as 14CO2. Although less than 5% of the dose was retained in tissues, approximately 12% was bound to the erythrocyte fraction of the blood. Following oral administration of [14C]BCB (80 mg/kg, 100 microgramsCi/kg), approximately 85% of the dose was absorbed, whereas 72% of the dosed radioactivity was recovered in the urine and 9.7% in the feces, 7.5% was exhaled as 14CO2, 3.5% bound to tissues, and 2. 6% bound to blood. Although parent compound could not be detected in the blood following oral administration, 2-MGN was detected (Cmax 0. 32 micrograms/ml). Following topical application of [14C]BCB (25 mg/kg, 50 microgramsCi/kg), less than 12% of the dose was absorbed, with the major route of excretion being the urine (6.6% of dose). Urinary metabolite profiles were nearly identical for each route of administration, and the primary urinary metabolite was a mercapturate conjugate of 2-MGN that was identified as N-acetyl-S-(2, 4-dicyanobutane)-L-cysteine. BCB was found to be extremely labile in whole blood, plasma, and glutathione containing solutions, and in each case the formation of 2-MGN could be reduced by the alkylation of free-sulfhydryls with N-ethylmaleimide. These results suggest that BCB is totally debrominated prior to systemic distribution, and tissue exposure to intact BCB seems to be exceedingly low regardless of route of administration.

Animals↗
PubMed1998

Sulfhydryl-dependent biotransformation and macromolecular binding of 1,2-dibromo-2,4-dicyanobutane in blood.

1,2-Dibromo-2,4-dicyanobutane (BCB) is a broad-spectrum microbicide used commercially in consumer products. The objectives of this study were to elucidate the biotransformation of BCB, characterize its ability to covalently bind macromolecules, and predict the possible toxicological ramifications of such events. After iv administration of [14C]BCB to male Fischer 344 rats, 14C-equivalents were observed to bind gradually to blood constituents. By 48 hr, approximately 12% of the total dose was covalently bound. At no time was parent compound detected in the blood. However, the debrominated BCB metabolite 2-methyleneglutaronitrile (MGN) was observed. In vitro experiments revealed that BCB was extremely labile and was readily debrominated in fresh whole blood, erythrocyte preparations, and buffered glutathione (GSH) solutions. In each case, the formation of MGN was inhibited by the alkylation of free sulfhydryls with N-ethylmaleimide (NEM). For every 1 mol of BCB converted to MGN, 2 mol of GSH were oxidized to glutathione disulfide (GSSG) (BCB + 2 GSH --> MGN + GSSG + 2 HBr). The oxidation of free sulfhydryls during the conversion of BCB to MGN caused erythrocyte hemolysis (EC50 approximately 1 mM) in isolated preparations. Hemolysis was increased by coincubation of BCB with NEM (EC50 approximately 0.3 mM) and was decreased by coincubation with GSH (EC50 > 3 mM). However, MGN did not cause hemolysis of erythrocytes, even at concentrations 10-fold higher than the EC50 of BCB. In vitro experiments also demonstrated that incubation with either BCB or MGN resulted in significant macromolecular binding to the erythrocyte fraction of the blood (approximately 80%). Incubation with NEM resulted in a significant decrease in binding for both BCB (11.3% bound) and MGN (29.5% bound). Because BCB is rapidly debrominated in whole blood, it appears that MGN is the reactive species responsible for macromolecular binding. From these studies, we conclude that the conversion of BCB to MGN is mediated by a free sulfhydryl-dependent biotransformation pathway. Furthermore, BCB biotransformation is required for erythrocyte binding, and the consumption of free sulfhydryls associated with the biotransformation of BCB is responsible for hemolysis.

Animals↗
PubMed1997

Suppression of Kupffer cell function prevents cadmium induced hepatocellular necrosis in the male Sprague-Dawley rat.

Exposure of humans to toxic metals and metalloids is a major environmental problem. Many metals, such as cadmium, can be hepatotoxic. However, the mechanisms by which metals cause acute hepatic injury are in many cases unknown. Previous reports suggest a major role for inflammation in acute cadmium induced hepatotoxicity. In initial experiments we found that a non-hepatotoxic dose of cadmium chloride (CdCl2; 2.0 mg/kg, i.v.) markedly increased the clearance rate of colloidal carbon from the blood, which is indicative of enhanced phagocytic activity by Kupffer cells (resident hepatic macrophages). Thus. the objective these studies was to determine the involvement of Kupffer cells in cadmium induced liver injury by inhibiting their function with gadolinium chloride (GdCl3). Male Sprague-Dawley rats were administered GdCl3 (10 mg/kg, i.v.) followed 24 h later by a single dose of CdCl2 (3.0 and 4.0 mg/kg, i.v.). Twenty four hours after CdCl2 administration animals were killed and the degree of liver toxicity was assessed using plasma alanine aminotransferase (ALT), as well as light microscopy. Cadmium chloride administration produced multifocal hepatocellular necrosis and increased plasma ALT activity. Pretreatment with GdCl3 significantly reduced both the morphological changes and hepatic ALT release caused by CdCl2. However, the protection was specific to the liver, and did not alter CdCl2 induced testicular injury, as determined by histopathological damage. In many cases, the inducible cadmium-binding protein, metallothionein (MT) is often an essential aspect of the acquisition of cadmium tolerance in the liver. Although cadmium caused a dramatic induction of hepatic MT (32-fold), GdCl3 caused only a minor increase (2-fold). Combined CdCl2 and GdCl3 treatment did not induce levels to an extent greater than CdCl2 alone. As expected, GdCl3 also caused a slight increase in the amount of cadmium associated with the liver. In cultured hepatocytes isolated from GdCl3 pretreated rats, CdCl2 induced cytotoxicity was not significantly altered compared to control hepatocytes, indicating that the mechanism of tolerance required the presence of other cell types. Thus, GdCl3 attenuation of CdCl2 induced hepatotoxicity does not appear to be caused by increased tissue MT content or a decreased susceptibility of hepatocytes to cadmium. From these data, we concluded that tolerance to cadmium induced hepatotoxicity involves the inhibition of Kupffer cell function which results in a decreased inflammatory response and an altered progression of hepatic injury. These data further indicate that Kupffer cell function is critical to cadmium induced hepatocellular necrosis.

Alanine Transaminase↗
PubMed1997

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.

Analysis of Variance↗
PubMed1997

Simultaneous determination of cyclohexene oxide and its metabolites in rat plasma and urine by gas chromatography.

An assay was developed for the simultaneous measurement of cyclohexene oxide and its metabolites (cyclohexanol, trans-cyclohexane-1,2-diol, cyclohexane-1,2-diol-O-glucuronide, and N-acetyl-S-(2-hydroxycyclohexyl)-L-cysteine) in rat urine and plasma using gas chromatography. A mixture of ethyl acetate-acetonitrile (70:30) was used as the extracting solvent for both matrices. This liquid-liquid extraction procedure was followed by the separation of cyclohexene oxide and its metabolites on an HP-FFAP fused-silica capillary column. In order to determine the amount of cyclohexane-1,2-diol-O-glucuronide, samples were incubated at 37 degrees C with beta-glucuronidase and the amount of cyclohexane-1,2-diol formed from the reaction determined. The extraction efficiencies of cyclohexene oxide and cyclohexanol were greater than 90% both in urine and plasma. However, recovery from the plasma and urine for trans-cyclohexane-1,2-diol (60-68%) and N-acetyl-S-(2-hydroxycyclohexyl)-L-cysteine (approximately 76%) were considerably less. Long term stability studies showed that urine samples spiked with cyclohexene oxide and trans-cyclohexane-1,2-diol are stable at -20 degrees C for up to 9 weeks. However, plasma samples are only stable for up to 2 weeks under the same conditions. The calibration curves for all analytes were linear over the range of 12.5 to 400 micrograms/ml and correlation coefficients (r2) were greater than 0.990. The limit of detection for cyclohexene oxide, cyclohexanol, and N-acetyl-S-(2-hydroxycyclohexyl)-L-cysteine is 1.56 micrograms/ml, while the limit of detection for trans-cyclohexane-1,2-diol is 3.12 micrograms/ml. This method has been used for the determination of the disposition and metabolism of cyclohexene oxide, and may be applied in environmental monitoring, as well as in microbiological studies for other epoxide materials.

Administration, Oral↗
PubMed1997

Tolerance induced by all-trans-retinol to the hepatotoxic effects of cadmium in rats: role of metallothionein expression.

Recently, it has been shown that large doses of all-trans-retinol (vitamin A) can potentiate the hepatotoxicity of several organic chemicals in the rat. Whether retinol pretreatment can alter the acute hepatotoxicity of an inorganic chemical, such as cadmium, is unknown. Therefore, the objective of this study was to determine how retinol might affect the acute toxicity of cadmium chloride (CdCl2) and to elucidate possible mechanisms. Cadmium exposure can induce acute, lethal hepatocellular necrosis in rodents, as well as lesions in the lung, kidney, testis, and gastrointestinal tract. In the present studies, male Sprague-Dawley rats were pretreated with retinol (75 mg/kg/day, po) for 7 consecutive days. One day after the last dose of retinol, animals were given a single injection of CdCl2 (2.5 to 4.0 mg/kg, iv). Cadmium chloride administration to unpretreated control rats caused extensive hepatic, renal, pulmonary, and testicular toxicity at 6, 24, and 48 hr postdosing as evaluated by plasma enzymes and/or histopathology. In retinol-pretreated rats, a significant attenuation of CdCl2-induced tissue injury was observed. Since the inducible cadmium-binding protein metallothionein (MT) is often an essential aspect of cadmium tolerance, its content in tissue was assessed using the cadmium-hemoglobin assay. Interestingly, retinol pretreatment significantly increased MT in the liver by sevenfold, but had no effect on lung, kidney, testicular, or pancreatic MT content. Although this increase in hepatic MT was much less than that induced by CdCl2, it was additive to the induction of CdCl2. Furthermore, the tissue distribution of cadmium was significantly altered by retinol pretreatment. The liver accumulated more cadmium, while less cadmium was found in the lung, kidney, and testis in retinol-pretreated rats than in controls. In monolayers of primary isolated hepatocytes, CdCl2-induced toxicity was significantly reduced in cells isolated from retinol-pretreated rats compared to those isolated from control rats. The dose response was shifted to the right and the in vitro cadmium LC50 was increased by in vivo retinol exposure from 1.1 +/- 0.1 to 2.4 +/- 0.04 microM. From these data it is concluded that the induction of hepatic MT is an essential aspect of retinol-induced tolerance to CdCl2 hepatotoxicity, as well as toxicity in other tissues.

Animals↗
PubMed1997

Reactions of peroxynitrite with gamma-tocopherol.

The reaction of peroxynitrite with gamma-tocopherol (gamma-TH) in a methanol/potassium phosphate buffer solution results in the formation of four major products, which were identified as 2,7,8-trimethyl-2-(4,8,12-trimethyldecyl)-5-nitro-6-chromanol++ + (NGT), 2,7,8-trimethyl-2-(4,8,12-trimethyldecyl)-5,6-chromaquinone (tocored), and two diastereomers of 8a-(hydroxy)-gamma-tocopherone. NGT was the major product formed in these reactions, and its formation was modestly increased by increasing amounts of Fe(3+)-EDTA. Tocored and NGT also were formed when gamma-TH was exposed to 3-morpholinosydnonimine (SIN-1), a compound that decomposes to form peroxynitrite. When gamma-TH reacted with the nitrating agent NO2+BF4- in acetonitrile or methanol/potassium phosphate buffer, NGT and tocored also were formed, but the major product detected was gamma-tocopherol quinone (gamma-TQ). This product was not detected in reactions involving peroxynitrite. Oxidation of gamma-TH by peroxynitrite involves nitration and electron transfer reactions. Since the product distribution in oxidations with NO2+BF4- differed substantially from that in oxidations with peroxynitrite and SIN-1, NO2+ appeared not to be the principal species involved in NGT formation. Nitration of gamma-TH may involve either peroxynitrite or some peroxynitrite-derived oxidant other than NO2+. Because of its stability and formation as a novel product of the reaction between gamma-TH with peroxynitrite, NGT may be a useful in vivo marker for peroxynitrite interactions with lipid structures that contain gamma-TH.

Magnetic Resonance Spectroscopy↗
PubMed1997

Modulation of Kupffer cell and peripheral blood monocyte activity by in vivo treatment of rats with all-trans-retinol.

Previous studies have shown that large doses of vitamin A potentiate chemical-induced liver injury and that the Kupffer cell is directly involved in this potentiation. Therefore, these studies were designed to determine if Kupffer cells isolated from vitamin A treated male Sprague-Dawley rats (75 mg/kg/day for 3-7 days as all- trans-retinol) had altered activity and function. Respiratory activity of Kupffer cells isolated from rats treated with vitamin A for 3 to 7 days markedly increased. Similarly, phagocytic activity was significantly elevated (up to 9-fold) after exposure to vitamin A for 3 to 7 days. Production of reactive oxygen species, measured by luminol-enhanced chemiluminescence of Kupffer cells isolated after 7 days of vitamin A exposure, was significantly higher than that of control cells when stimulated with opsonized zymosan. Also, the release of superoxide anion by individual Kupffer cells isolated from vitamin A treated rats was nearly three times greater than that of control cells. Basal production of tumor necrosis factor-alpha (TNF-alpha) and prostaglandin E2 (PGE2) production were significantly elevated in Kupffer cells isolated from rats treated with vitamin A. Lastly, peripheral blood monocytes (PBMC) isolated from rats treated with vitamin A for 7 days had a significantly greater respiratory activity, as well as TNF-alpha and PGE2 production, than PBMC isolated from control rats. Our data suggest that large doses of vitamin A enhance both Kupffer cell and PBMC function. Upregulation of the activity by these phagocytic cells may play a role in the vitamin A potentiation of chemical-induced liver injury.

Animals↗
PubMed1997

Absorption, disposition kinetics, and metabolic pathways of cyclohexene oxide in the male Fischer 344 rat and female B6C3F1 mouse.

Cyclohexene oxide (CHO) is a monomer intermediate used in the synthesis of pesticides, pharmaceuticals, and perfumes. Although CHO has a variety of industrial uses where direct human exposure is possible, very little is known about its fate in the body. Therefore, the objectives of this study were to determine the absorption, distribution, metabolism, and excretion of cyclohexene oxide after oral, intravenous, and dermal exposure in male Fischer 344 rats and female B6C3F, mice. After intravenous administration of [14C]CHO (50 mg/kg), CHO was rapidly distributed, metabolized, and excreted into the urine. Plasma concentrations of CHO rapidly declined and were below the limit of detection within 60 min. Average (+/- SD) values for terminal disposition half-life, apparent volume of distribution at steady-state, and systemic body clearance were: 19.3 +/- 1.6 min; 0.44 +/- 0.08 liter/kg; and 31.3 +/- 0.5 ml/kg * min, respectively. After oral administration of [14C]CHO (10 and 100 mg/kg), it was found that 14C-equivalents were rapidly excreted in the urine of both species. At 48 hr, the majority of the dose (73-93%) was recovered in urine, whereas fecal elimination accounted for only 2-5% of the dose. At no time after oral administration was parent CHO detected in the blood. However, its primary metabolite cyclohexane-1,2-diol was present for different lengths of time depending on the dose. Four metabolites were detected and identified in mouse urine by MS: cyclohexane-1,2-diol; cyclohexane-1,2-diol-O-glucuronide; N-acetyl-S-(2-hydroxycyclohexyl)-L-cysteine; and cyclohexane-1,2-diol-O-sulfate. The sulfate conjugate was not present in rat urine. Topical application of [14C]CHO (60 mg/kg) provided poor absorption in both species. The majority of 14C-equivalents applied dermally were recovered from the charcoal skin trap (approximately 90% of the dose). Only 4% of the dose was absorbed, and the major route of elimination was via the urine. To evaluate the toxicity of CHO, animals were given daily doses of CHO orally and topically for 28 days. No statistically significant changes in final body weights or relative organ weights were noted in rats or mice treated orally with CHO up to 100 mg/kg or up to 60 mg/kg when given topically. Very few lesions were found at necropsy, and none were considered compound related. In conclusion, regardless of route, CHO is rapidly eliminated and excreted into the urine. Furthermore, after either oral or dermal administration, it is unlikely that CHO reaches the systemic circulation intact due to its rapid metabolism, and is therefore unable to cause toxicity in the whole animal under the test conditions used in this study.

Administration, Cutaneous↗
PubMed1997

Oral and topical absorption, disposition kinetics, and the metabolic fate of trans-methyl styryl ketone in the male Fischer 344 rat.

trans-Methyl styryl ketone (MSK; trans-4-phenyl-3-buten-2-one) is a beta-unsaturated ketone that has a wide range of uses in industry and is present in numerous consumer products. Although MSK has been shown to be positive in several in vitro mutagenic assays, it does not seem to be overtly toxic in animal models. This lack of toxicity may relate to its poor absorption and/or rapid elimination. However, little is known about the fate of MSK in the body. Studies were conducted to characterize the absorption, and disposition kinetics of MSK after intravenous, oral, and topical administration to male Fischer 344 rats. After intravenous administration of [14C]MSK (20 mg/kg, 120 microCi/kg), blood concentration-time data could be characterized with a biexponential equation and apparent first-order elimination kinetics. The following pharmacokinetic parameter values were obtained (mean +/- SD): terminal disposition half-life, 17.7 +/- 0.08 min; apparent steady-state volume of distribution, 0.89 +/- 0.14 liters/kg; systemic body clearance, 68.9 +/- 10.0 ml/kg *min; and mean residence time, 13.1 +/- 2.2 min. Within 48 hr, 95.5% of the dose was excreted in the urine and 2.7% in the feces. The major blood metabolite after intravenous administration was identified by GC/MS as the 4-phenyl-3-buten-2-ol (methyl styryl carbinol). After oral administration of [14C]MSK (200 mg/kg, 100 microCI/kg), approximately 96.6% of the dosed radioactivity was recovered in the urine and 4.8% in the faces within 48 hr. Major urinary metabolites identified by LC-MS/MS and quantified by HPLC radioassay were N-phenylacetyl-L-glycine (64.9% of dose) and N-benzyl-L-glycine (9.9% of dose). Parent compound could not be detected in the blood after oral administration, and 14C-equivalents in the blood never exceeded 1.3% of the dose. Results suggest near-total presystemic elimination of the oral dose. After topical application of [14C]MSK (250 mg/kg, 50 microCi/kg), > 60% of the dose was absorbed, and the majority of the dose was excreted into the urine (55% of dose) in the form of metabolites. Urinary metabolites were similar to those described after oral administration. 14C-equivalents were not detected in the blood at any time after topical administration. These results indicate that MSK is almost totally metabolized before systemic distribution after oral or topical administration. The systemic exposure dose of MSK seems to be exceedingly low at the doses studied herein.

Administration, Oral↗