Comparative DNA cross-linking by activated pyrrolizidine alkaloids.

The toxicity and bioactivity of pyrrolizidine alkaloids (PAs), common constituents of hundreds of plant species, and in herbal remedies and folk medicines prepared thereof, are probably due to their ability to form DNA cross-linking. We investigated DNA cross-linking activity by chemically-activated PAs from four different structural classes in Madin-Darby bovine kidney (MDBK) cells and in pBR322 DNA. In cell culture, alpha,beta-unsaturated macrocyclic diester pyrroles dehydrosenecionine (DHSN), dehydroriddelliine (DHRD) and the saturated macrocyclic diester pyrrole dehydromonocrotaline (DHMO) were significantly more potent cross-linkers than the simple necine base (retronecine) and an N-oxide (indicine N-oxide; INO) as determined by alkaline elution. The proportion of total DNA cross-links that were proteinase K-resistant (DNA-DNA cross-links) induced by the various pyrroles ranged from 0.08 (DHRN) to 0.67 (DHSN). Those pyrroles that were potent cross-linkers of cellular DNA also cross-linked, in a dose-dependent manner, Bam HI-digested pBR322 DNA as assessed by a gel retardation assay. The possible functional relevance of pyrrole-DNA cross-links was determined by their ability to interrupt PCR amplification of a 1.129 kb segment of pBR322. Dehydrosenecionine completely inhibited amplification, while DHMO was of intermediate potency, while DHRN and INO had no effect. Taken together, these studies suggest that structural features, most notably the presence of a macrocyclic diester, confer potent cross-link activity to PAs. In any event, DNA-DNA cross-linking is probably biologically relevant as indicated by their interference with DNA replication.     

Ethanol enhancement of cocaine-induced hepatotoxicity

Ethanol administration (4.3% ethanol in a liquid diet for 5 days) to adult male mice produced a peak blood ethanol concentration of 180 mg/100 ml and resulted in a significant increase in hepatic cytochrome P-450 levels. Ethanol treatment significantly reduced cocaine-induced acute lethality from 67 to 23 per cent. However, ethanol treatment resulted in a potentiation of a latent (1–7 day) cocaine-induced toxicity characterized by hepatic dysfunction, as monitored by serum glutamate-pyruvate transaminase (SGPT) activity, and a profound centrilobular necrosis. The minimum dose of cocaine that caused elevations of SGPT activity was 20 mg/kg, i.p.; maximum elevations of SGPT activity were produced by a dose of 40 mg/kg, i.p. The peak elevations of SGPT activity were seen between 24 and 30 hr following adminstration of cocaine. Frank hepatic necrosis was seen following administration of 30 mg/kg of cocaine. Ethanol potentiation of cocaine-induced hepatoxicity was dependent on induction of the hepatic cytochrome P-450 mixed function oxidase enzyme system. The intralobular location of the cocaine-induced hepatic necrosis was also dependent upon the inducing agent used. Ethanol potentiated the cocaine-induced delayed toxicity presumably by enhancing its biotransformation to a chemically reactive intermediate metabolite that produced the hepatic centrilobular necrosis.     

Tissue thiol induction in mice and protective effect against pyrrolizidine alkaloids by dietary ethoxyquin and methionine hydroxy analog

Dietary administration of ethoxyquin (EQ) and methionine hydroxy analog (MHA) induced the thiol concentrations in liver, kidney, small intestine and stomach of mice accompanied with hepatic hypertrophy. The hepatic thiol levels among EQ, EQ-MHA, or EQ-methionine treated mice were significantly higher (p less than 0.01) than that of the controls. Hepatic hypertrophy was apparent with 0.031% EQ-0.063% MHA in the diet. EQ feeding, as 0.5% w/w of feed with or without another additive, also resulted in reduced weight gain. The toxicity of pyrrolizidine alkaloids (PAs) was reduced by feeding diets containing 0.063% EQ-0.125% MHA or 0.125% EQ-0.25% MHA. The intraperitoneal LD50 values of PAs in these two EQ-MHA feeding groups were 92.8 and 94.0 mg/kg, respectively, compared to that of 71.3 mg/kg for the controls.     

Acetaminophen-induced hepatic glycogen depletion and hyperglycemia in mice

Two hours following administration of a hepatotoxic dose of acetaminophen (500 mg/kg, i.p.) to mice, liver sections stained with periodic acid Schiff reagent showed centrilobular hepatic glycogen depletion. A chemical assay revealed that following acetaminophen administration (500 mg/kg) hepatic glycogen was depleted by 65% at 1 hr and 80% at 2 hr, whereas glutathione was depleted by 65% at 0.5 hr and 80% at 1.5 hr. Maximal glycogen depletion (85% at 2.5 hr correlated with maximal hyperglycemia (267 mg/100 ml at 2.5hr). At 4.0 hr following acetaminophen administration, blood glucose levels were not significantly different from saline-treated animals; however, glycogen levels were still maximally depleted. A comparison of the dose-response curves for hepatic glycogen depletion and glutathione depletion showed that acetaminophen (50–500 mg/kg at 2.5 hr) depleted both glycogen and glutathione by similar percentages at each dose. Since acetaminophen (100 mg/kg at 2.5 hr) depleted glutathione and glycogen by approximately 30%, evidence for hepatotoxicity was examined at this dose to determine the potential importance of hepatic necrosis in glycogen depletion. Twenty-four hours following administration of acetaminophen (100 mg/kg) to mice, histological evidence of hepatic necrosis was not detected and serum glutamate pyruvate transaminase (SGPT) levels were not significantly different from saline-treated mice. The potential role of glycogen depletion in altering the acetaminophen-induced hepatotoxicity was examined subsequently. When mice were fasted overnight, hepatic glutathione and glycogen were decreased by 40 and 75%, respectively, and fasted animals showed a dramatic increase in susceptibility to acetaminophen-induced hepatotoxicity as measured by increased SGPT levels. Availability of glucose in the drinking water (5%) overnight resulted in glycogen levels similar to those in fed animals, whereas hepatic glutathione levels were not significantly different from those of fasted animals. Fasted animals and animals given glucose water overnight were equally susceptible to acetaminophen-induced hepatotoxicity, as quantitated by increases in SGPT levels 24 hr after drug administration. The potential role of a reactive metabolite in glycogen depletion was investigated by treating mice with N-acetylcysteine to increase detoxification of the reactive metabolite. N-Acetylcysteine treatment of mice prevented acetaminophen-induced glycogen depletion.     

Medicinal plants in China containing pyrrolizidine alkaloids

Medicinal plants and remedies are widely used for various ailments throughout the world. Many of these plants contain pyrrolizidine alkaloids (PAs) which are hepatotoxic, pneumotoxic, genotoxic, neurotoxic, and cytotoxic. As a result of their use in Traditional Chinese Medicine (TCM), medicinal plants are becoming increasingly important not only in China but also in many other countries. This paper will therefore give, a critical overview of PA-containing plants belonging mainly to the families Boraginaceae, Leguminosae (Tribus Crotalarieae), and Asteraceae (Tribus Senecioneae and Eupatorieae). The PAs contained in the 38 plants described here differ widely in their structure and toxicity. Their metabolism and the resulting toxicity will be discussed, the dehydroalkaloids (DHAlk) produced in the liver playing a key role in cases of intoxications.     

Synergistic hepatotoxicity from coexposure to bacterial endotoxin and the pyrrolizidine alkaloid monocrotaline

Individuals are commonly exposed to bacterial endotoxin (lipopolysaccharide [LPS]) through gram-negative bacterial infection and from its translocation from the gastrointestinal lumen into the circulation. Inasmuch as noninjurious doses of LPS augment the hepatotoxicity of certain xenobiotic agents, exposure to small amounts of LPS may be an important determinant of susceptibility to chemical intoxication. Monocrotaline (MCT) is a pyrrolizidine alkaloid phytotoxin that at large doses produces centrilobular liver lesions in rats. In the present study, MCT was coadministered with LPS to determine whether LPS would enhance its hepatotoxicity. Doses of MCT (100 mg/kg, ip) and LPS (7.4 x 10(6) EU/kg, iv), which were nonhepatotoxic when administered separately, produced significant liver injury in male, Sprague-Dawley rats when given in combination. Within 18 h after MCT administration, this cotreatment resulted in enhanced plasma alanine aminotransferase and aspartate aminotransferase activities, two markers of liver injury. Histologically, overt hemorrhage and necrosis appeared between 12 and 18 h. The lesions were centrilobular and midzonal and exhibited characteristics similar to lesions associated with larger doses of MCT and LPS, respectively. In the presence of LPS, the threshold for MCT toxicity was reduced to 13-33% of the dose required for toxicity with MCT alone. A study in isolated, hepatic parenchymal cells revealed no interaction between MCT and LPS in producing cytotoxicity. In summary, coexposure of rats to noninjurious doses of MCT and LPS resulted in pronounced liver injury. Results in vitro suggest that the enhanced toxicity does not result from a direct interaction of MCT and LPS with hepatic parenchymal cells. These results provide additional evidence that exposure to small amounts of LPS may be a determinant of susceptibility to food-borne hepatotoxins.     

Hepatotoxicity of butylated hydroxytoluene and its analogs in mice depleted of hepatic glutathione

Butylated hydroxytoluene (2,6-di-tert-butyl-4-methylphenol, BHT) has been reported to be a lung toxicant. Mice treated with BHT (200-800 mg/kg, po) in combination with an inhibitor of glutathione (GSH) synthesis, buthionine sulfoximine (BOS; 1 hr before and 2 hr after BHT, 4 mmol/kg per dose, ip) developed hepatotoxicity characterized by an increase in serum glutamic pyruvic transaminase (GPT) activity and centrilobular necrosis of hepatocytes. The hepatotoxic response was both time- and dose-dependent. BHT (up to 800 mg/kg) alone produced no evidence of liver injury. As judged by the observation of normal serum GPT, drug metabolism inhibitors such as SKF-525A, piperonyl butoxide, and carbon disulfide prevented the hepatotoxic effect of BHT given in combination with BSO. On the other hand, pretreatment with cedar wood oil resulted in increased hepatic injury in mice treated with both BHT and BSO. Pretreatment with phenobarbital also tended to increase hepatic injury as judged by changes in serum GPT. These results suggest that BHT is activated by a cytochrome-P-450-dependent metabolic reaction and that the hepatotoxic effect is caused by inadequate rates of detoxification of the reactive metabolite in mice depleted of hepatic GSH by BSO administration. The hepatotoxic potencies of BHT-related compounds also were examined in BSO-treated animals. For hepatotoxicity, the phenolic ring must have benzylic hydrogen atoms at the 4 position and an ortho-alkyl group(s) that moderately hinders the hydroxyl group. These structural requirements essentially are the same as those for the toxic potency in the lung (T. Mizutani, I. Ishida, K. Yamamoto, and K. Tajima (1982), 62, 273-281) and support the hypothesis that BHT-quinone methide plays a role in producing liver damage in mice with depressed hepatic GSH levels.     

S-Adenosylmethionine (SAMe) attenuates acetaminophen hepatotoxicity in C57BL/6 mice

Hepatic toxicity is associated with excessive dosages of the over the counter analgesic, acetaminophen (APAP). The aim of this study was to explore protection by the nutritional agent S-adenosylmethionine (SAMe) on APAP hepatotoxicity. Male C57BL/6 mice were injected intraperitoneal (i.p.) with 500 mg/kg (15 ml/kg) APAP or water vehicle (VEH). SAMe was injected i.p. at a dose of either 1000 mg/kg (5 ml/kg) just prior or 500 mg/kg SAMe 15 min prior to administration of VEH or APAP. Comparison of groups showed that SAMe reduced APAP toxicity. Plasma alanine aminotransferase (ALT) levels were increased 2 and 4 h after APAP administration when compared to vehicle (VEH) controls. Liver weight was increased relative to the VEH group within 4 h after APAP treatment. Histological examination by light microscopy confirmed small changes in morphology within 2 h after APAP injection and marked centrilobular necrosis within 4 h in the APAP group. In contrast, when APAP was administered to SAMe pretreated mice, ALT and liver weights were comparable to the VEH and SAMe groups. Histological examination also showed that SAMe produced a marked protection in APAP mediated centrilobular necrosis at 4 h after APAP injection. APAP administration depressed hepatic glutathione levels when monitored at 2 and 4 h. Lipid peroxidation was induced above VEH values 2 and 4 h after APAP injection. Consistent with the SAMe protection of APAP hepatic toxicity, the expected depletion of hepatic glutathione (GSH) levels by APAP was prevented by SAMe pretreatment. SAMe pretreatment also prevented the induction of lipid peroxidation at 2 and 4 h post-APAP administration. In conclusion, SAMe provides protection from APAP hepatic toxicity at 2 and 4 h post-APAP injection. SAMe pretreatment prevented APAP associated depletion in hepatic glutathione and induction of lipid peroxidation as part of its mechanism of protection.     

Exaggerated hepatotoxicity of acetaminophen in mice lacking tumor necrosis factor receptor-1. Potential role of inflammatory mediators

Transgenic mice with a targeted disruption of the tumor necrosis factor receptor 1 (TNFR1) gene were used to analyze the role of TNF-alpha in pro- and anti-inflammatory mediator production and liver injury induced by acetaminophen. Treatment of wild-type mice with acetaminophen (300 mg/kg) resulted in centrilobular hepatic necrosis. This was correlated with expression of inducible nitric oxide synthase (NOS II) and nitrotyrosine staining of the liver. Expression of macrophage chemotactic protein-1 (MCP-1), KC/gro, interleukin-1beta (IL-1beta), matrix metalloproteinase-9 (MMP-9), and connective tissue growth factor (CTGF), inflammatory mediators known to participate in tissue repair, as well as the anti-inflammatory cytokine, interleukin-10 (IL-10), also increased in the liver following acetaminophen administration. TNFR1(-/-) mice were found to be significantly more sensitive to the hepatotoxic effects of acetaminophen than wild-type mice. This was correlated with more rapid and prolonged induction of NOS II in the liver and changes in the pattern of nitrotyrosine staining. Acetaminophen-induced expression of MCP-1, IL-1beta, CTGF, and MMP-9 mRNA was also delayed or reduced in TNFR1(-/-) mice relative to wild-type mice. In contrast, increases in IL-10 were more rapid and more pronounced. These data demonstrate that signaling through TNFR1 is important in inflammatory mediator production and toxicity induced by acetaminophen.     

A nutrient mixture prevents acetaminophen hepatic and renal toxicity in ICR mice

Acetaminophen (APAP) overdose is often fatal, leading to fulminant hepatic and renal tubular necrosis in humans and animals. We studied the effect of a nutrient mixture (NM) containing, among other nutrients, lysine, proline, ascorbic acid, N-acetyl cysteine, and green tea extract, which has previously been demonstrated to exhibit a broad spectrum of therapeutic properties on APAP-induced hepatic and renal damage in ICR (Imprinting Control Region) mice. Seven-week-old male ICR mice were divided into four groups (A-D) of five animals each. Groups A and C mice were fed a regular diet for 2 weeks, while groups B and D mice were supplemented with 0.5% NM (w/w) during that period. Groups A and B received saline i.p., while groups C and D received APAP (600 mg/kg) i.p. All animals were killed 24 h after APAP administration, serum was collected to assess the liver and kidney functions, and the livers and kidneys were excised for histology. Mean serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, BUN (Blood Urea Nitrogen), creatinine, and BUN/creatinine ratios were comparable in groups A and B, increased markedly in group C and significantly lower in group D compared with group C. APAP caused significant centrilobular necrosis and glomerular damage in unsupplemented animals, while NM prevented these alterations. The results indicate that NM has potential to protect against APAP-induced liver and kidney damage.     

Hepatotoxicity of acetaminophen in neonatal and young rats. I. Age-related changes in susceptibility

The susceptibility of neonatal (11 days) and young rats (19 and 33 days) to acetaminophen-induced hepatic necrosis was examined. Acetaminophen-induced lethality (LD50) was slightly lower in 19-day-old animals (840 mg/kg) compared to 11- and 33-day-old animals (1220 and 1580 mg/kg, respectively). A toxic dose of the drug ( LD20 ) produced elevated serum glutamate-pyruvate transaminase and lactate dehydrogenase activities 20-24 hr after drug administration only in 19- and 33-day-old animals. Serum enzyme elevation was not observed after a toxic dose of acetaminophen ( LD20 or LD50) in 11-day-old rats. Histological evaluation showed that both 19- and 33-day-old rats developed extensive hepatic centrilobular damage, whereas morphological parameters in 11-day-old animals given acetaminophen were not different from controls. It appears that high doses of acetaminophen are lethal to young rats, but that 11-day-old animals are different from 19-day-old and older rats in that the neonatal animals lack susceptibility to the hepatotoxic effects of the drug. Lower susceptibility of the neonatal rat liver to the hepatic effects of two other hepatotoxicants (bromobenzene and tannic acid) was also observed.     

New pyrrolizidine alkaloids from Heliotropium crassifolium

Heliotropium crassifolium Boiss, (Boraginaceae) from a population of Ilam, western region of Iran was studied for pyrrolizidine alklaoids (PAs). Four alkaloids have been identified: europine 1, europine N-oxide 2 and a new pyrrolizidine alkaloids ilamine 3 and its N-oxide 4, respectively. Their structures were elucidated by IR, 1H-NMR and EIMS data.     

Hepatotoxicity and pulmonary toxicity of pennyroyal oil and its constituent terpenes in the mouse

Pennyroyal oil, an aromatic mint-like oil used as a flavoring and fragrance agent and as a herbal medicine, caused acute hepatic and lung damage at doses of 400 mg/kg, ip, and higher in male Swiss-Webster mice. Cellular necrosis was localized to the centrilobular regions of the liver and bronchiolar epithelial cells of the lung. Capillary gas chromatographic analysis of samples of pennyroyal oil that were obtained from health food stores showed the presence of several monoterpene constituents. R-(+)-Pulegone was the major terpene and constituted greater than 80% of the constituent terpenes in the oils that were examined. Pulegone and two other constituent terpenes, isopulegone and menthofuran, were found to be both hepatotoxic and lung toxic. Based on results of histologic scoring of necrosis, plasma GPT elevations, and hepatic glutathione depletion, R-(+)-pulegone is the terpene primarily responsible for the tissue necrosis. Furthermore, results of toxicity tests with several congeners of R-(+)-pulegone, including the enantiomeric S-(?)-pulegone, strongly implicated the α-isopropylidene ketone group as the structural unit required for eliciting hepatotoxicity, although the configurational orientation of the methyl group can modulate the hepatotoxic response.     

Thirteen-week oral toxicity of 1,4-dioxane in rats and mice

Subchronic oral toxicity of 1,4-dioxane was examined by administering 1,4-dioxane in drinking water at 6 different concentrations of 0 (control), 640, 1,600, 4,000, 10,000 or 25,000 ppm (wt/wt) to F344 rats and BDF(1)mice of both sexes for 13 weeks. Food and water consumption and terminal body weight were decreased dose-dependently in rats and mice. A dose-dependent increase in the relative weights of kidney and lung was noted in rats and mice, while the relative liver weight was increased only in rats. Increases in plasma levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and a decrease in plasma glucose were noted primarily in the rats and mice dosed 25,000 ppm. Histopathological examination revealed that 1,4-dioxane affected the upper and lower respiratory tracts, liver, kidneys and brain in rats, while only the former two organs were affected in mice. Nuclear enlargement occurred in the respiratory, olfactory, tracheal and bronchial epithelia of the 1,4-dioxane-dosed rats and mice. The 1,4-dioxane-induced hepatic lesions were characterized by centrilobular swelling and necrosis in rats and mice and by glutathione S-transferase placental form (GST-P)-positive altered hepatocellular foci in rats, which are known as preneoplastic lesions. A no-observed-adverse-effect-level (NOAEL) was determined at 640 ppm for both rats and mice, since the nuclear enlargement in the nasal respiratory epithelium and the centrilobular swelling of hepatocytes in rats and the nuclear enlargement in the bronchial epithelium in mice were observed at 1,600 ppm. The NOAEL value corresponded to the estimated 1,4-dioxane intake of 52 mg/kg/day in rats and 170 mg/kg/day in mice.     

Species differences in the hepatic microsomal enzyme metabolism of the pyrrolizidine alkaloids

Species differences in pyrrolic metabolites and senecionine (SN) N-oxide formation among eight animal species (sheep, cattle, gerbils, rabbits, hamsters, Japanese quail, chickens, rats) varying in susceptibility to pyrrolizidine alkaloid (PA) intoxication were measured in vitro by hepatic microsomal incubations. The results suggested that there is not a strong correlation between the production of pyrrolic metabolites and susceptibility of animals to PA toxicity. The rate of PA activation in hamsters, a resistant species, measured by formation of (±)6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) far exceeded the rate of SN N-oxide formation (detoxification) (DHP/N-oxide=2.29). In contrast, SN N-oxide was the major metabolite in sheep, another resistant species, with much lower production of DHP (DHP/N-oxide=0.26). The roles of cytochrome P450s and flavin-containing monooxygenases (FMO) in bioactivation and detoxification of pyrrolizidine alkaloids (PA) were studied in vitro using sheep and hamster hepatic microsomes. Chemical and immunochemical inhibition data suggested that the conversion of SN to DHP is catalyzed mainly by cytochrome P450s (68–82%), whereas the formation of SN N-oxide is carried out largely by FMO (55–71%). There also appeared to be a high rate of glutathione–DHP conjugation in hamster (63%) and sheep (79%) liver microsomal incubation mixtures. Therefore, low rates of pyrrole metabolite production coupled with glutathione conjugation in sheep may explain the resistance of sheep to SN, whereas the high rate of GSH-DHP conjugation may be one of the factors contributing to the resistance of hamsters to intoxication by this PA.     

Synergistic effects of pyrrolizidine alkaloids and lipopolysaccharide on preterm delivery and intrauterine fetal death in mice

Preterm birth is the leading cause of death for newborn infants, and lipopolysaccharide (LPS) is commonly used to induce preterm delivery in experimental animals. Pyrrolizidine alkaloids (PAs) are widespread and occur in foods, herbs, and other plants. This study was to investigate the synergistic effects of LPS and two representative PAs, retrorsine (RTS) and monocrotaline (MCT), on preterm delivery and fetal death. Pregnant Kunming mice were divided into seven groups: control, RTS, MCT, LPS, RTS + LPS and two MCT + LPS groups. Animals in PAs and PAs + LPS groups were dosed intragastrically with RTS (10 mg/kg) or MCT (20 mg/kg or 60 mg/kg) from gestational day (GD) 9 to GD16; mice given LPS were injected intraperitoneally with 150 μg/kg on GD15.5. Latencies to delivery, numbers of pups live and dead at birth were recorded, and livers of live neonates were collected. The incidence of LPS-induced preterm birth was enhanced in dams pretreated with MCT, and combination of PAs and LPS increased fetal mortality from PAs. The enhancement of LPS-induced preterm delivery and fetal demise in animals exposed chronically to PAs and other substances found in foods and beverages consumed widely by humans merits further focused investigation.     

A preliminary assessment of the acute and subchronic toxicity profile of phase2: an alpha-amylase inhibitor

Phase2, which has been reported to reduce body weight by its inhibition of a-amylase, was evaluated for toxicity in young adult male and female Wistar rats (10 animals/dose group). Evaluations included mortality, change in body weight, food consumption pattern, organ weight, and other adverse side reactions as well as hematological, biochemical, and histopathological analyses. Acute toxicity was determined after a single dose of Phase2 by oral gavage at doses of 5.0, 1.0, and 0.5 g/kg body weight. Animals were sacrificed on fourteen days after Phase2 administration. Subchronic toxicity was determined by administering Phase2 daily for 90 days to rats, at doses of 1.0, 0.5, and 0.2 g/kg body weight. These animals were sacrificed on day 90. Acute and subchronic administration of Phase2 did not produce any adverse reactions or any significant change in the loss of body weight as compared to untreated controls, organ weight, and mortality. Administration of Phase2 did not alter the hepatic and renal function, and did not produce any change in the hematological parameters and in lipid profile. Subchronic administration produced a reduction in the food consumption after 77 days (1.0 g/kg body weight). These data indicate that acute and subchronic administration of Phase2 did not produce any toxicity to rats as evident from weight change, mortality, and limited biochemical and histopathological analyses.     

Endothelial cell injury and fibrin deposition in rat liver after monocrotaline exposure.

Monocrotaline (MCT) is a pyrrolizidine alkaloid (PA) plant toxin that produces hepatotoxicity in people and animals. Human exposure to PAs occurs through consumption of contaminated grains and herbal remedies. Injection (ip) of MCT in rats produced dose-dependent hepatic parenchymal cell injury that was significant at 200 mg/kg. Injection of 300 mg/kg MCT produced time-dependent hepatotoxicity with significant injury beginning by 12 h after treatment. Histopathologic examination of liver sections revealed coagulative hepatocellular necrosis, widening of sinusoids and hemorrhage in centrilobular regions. MCT-induced damage to central venular endothelial cells (CVECs) and sinusoidal endothelial cells (SECs) in the liver was quantified using immunohistochemical staining and by increased plasma hyaluronic acid concentration. MCT damaged CVECs and SECs in the liver by 8 h after treatment. Extensive endothelial cell injury was restricted to centrilobular regions. To determine if damage to endothelial cells in the liver stimulated activation of the coagulation system, fibrin deposition was quantified using immunohistochemistry. Extensive fibrin deposition occurred in the liver after MCT treatment and was restricted to centrilobular regions. Interestingly, both endothelial cell damage and fibrin deposition preceded the onset of hepatic parenchymal cell injury. These results suggest that endothelial cell damage and fibrin deposition in centrilobular regions of the liver are prominent features of MCT-induced liver injury.     

Formaldehyde and hepatotoxicity: a review

Exposure to formaldehyde appears to be associated with hepatoxicity in many species, including humans, following injection, ingestion, or inhalation. Macroscopic, microscopic, and biochemical manifestations in the liver include alterations in weight, centrilobular vacuolization, focal cellular necrosis, and increased alkaline phosphatase concentrations. Time-related changes in the pattern of the effects are suggested as one goes from acute exposure by inhalation at greater concentrations to repeated exposure at lesser concentrations. Although the hepatic changes are generally not extensive and can be reversible following acute exposure, the potential exists for them to progressively become more serious with repeated exposures. There are several possible mechanisms for the toxicity. Depending on the route of exposure could include direct effects on hepatocytes and/or indirect effects through the circulatory and immune systems. The catabolism of formaldehyde includes conversion to CO2 by reactions involving glutathione. Many hepatotoxic chemicals require glutathione for detoxification. Formaldehyde may then have the potential to cause additive toxicity with such chemicals in some circumstances.     

Riddelliine N-oxide is a phytochemical and mammalian metabolite with genotoxic activity that is comparable to the parent pyrrolizidine alkaloid riddelliine

Pyrrolizidine alkaloids (PAs) and their N-oxide derivatives are naturally-formed genotoxic phytochemicals that are widely distributed throughout the world. Although, the quantities of PAs and PA N-oxides in plants are nearly equal, the biological and genotoxic activities of PA N-oxides have not been studied extensively. PA N-oxides are major metabolites of PAs and are generally regarded as detoxification products. However, in this study, we report that rat liver microsomes converted riddelliine N-oxide to the genotoxic 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) metabolite. Metabolism of riddelliine N-oxide by rat liver microsomes under hypoxic conditions (argon) generated predominantly the parent PA, riddelliine. The reduction of riddelliine N-oxide to riddelliine was diminished, when the metabolism of riddelliine N-oxide with rat liver microsomes was conducted aerobically. Rat liver microsomal incubations of riddelliine N-oxide in the presence of calf thymus DNA produced a set of DHP-derived DNA adducts as detected and quantified by 32P-postlabeling/HPLC. The same DHP-derived DNA adducts were also found in liver DNA of F344 rats fed riddelliine N-oxide or riddelliine. When rats received doses of 1.0 mg/kg riddelliine N-oxide for three consecutive days, the level of DNA adducts was 39.9 +/- 0.6 adducts/10(7) nucleotides, which was 2.6-fold less than that measured in rats treated with riddelliine at the same dose. We have previously shown that these DHP-derived DNA adducts are produced by chronic feeding of riddelliine and that the adduct levels correlated with liver tumor formation. Results presented in this paper indicate that riddelliine N-oxide, through its conversion to riddelliine, is also a potential genotoxic hepatocarcinogen.