Safety assessment of pomegranate fruit extract: acute and subchronic toxicity studies.

Pomegranate (Punica granatum L.) fruit is widely consumed as fresh fruit and juice. Because of its potential for health benefits, pomegranate fruit extracts have been commonly marketed as dietary supplements in recent years. The objective of the present study was to investigate potential adverse effects, if any, of a standardized pomegranate fruit extract in rats following subchronic administration. The extract was standardized to 30% punicalagins, the active anomeric ellagitannins responsible for over 50% of the antioxidant potential of the juice. The oral LD(50) of the extract in rats and mice was found to be greater than 5 g/kg body weight. The intraperitoneal LD(50) in rats and mice was determined as 217 and 187 mg/kg body weight, respectively. In the subchronic study, Wistar strain rats (10/sex/group) were administered via gavage 0 (control), 60, 240 and 600 mg/kg body weight/day of the extract for 90 days. Two additional groups received 0 and 600 mg/kg/day of the extract for 90 days, followed by a 28 day recovery phase. Compared to the control group, administration of the extract did not result in any toxicologically significant treatment-related changes in clinical observations, ophthalmic examinations, body weights, body weight gains, feed consumption, clinical pathology evaluations and organ weights. The hematology and serum chemistry parameters that showed statistical significant changes compared to control group were within the normal laboratory limits and were considered as biological variations and not the toxic effect of the extract. Terminal necropsy did not reveal any treatment-related gross or histopathology findings. Based on the results of this study, the no observed-adverse-effect level (NOAEL) for this standardized pomegranate fruit extract was determined as 600 mg/kg body weight/day, the highest dose tested.

Safety assessment of astaxanthin-rich microalgae biomass: Acute and subchronic toxicity studies in rats.

Astaxanthin, a natural nutritional component, is marketed as a dietary supplement around the world. The primary commercial source for astaxanthin is Haematococcus pluvialis (microalgae). The objective of the present study was to investigate the acute and subchronic toxicity of an astaxanthin-rich biomass of H. pluvialis (AstaCarox). The oral LD(50) of the biomass in rats was greater than 12g/kg body weight. In the subchronic study, Wistar rats (10/sex/group) were fed diets containing 0%, 1%, 5% and 20% of the biomass (weight/weight) for 90 days. trans-Astaxanthin was quantifiable in the plasma of the high-dose treated group only. Compared to the control group, no treatment-related biologically significant effects of astaxanthin were noted on body weight or body weight gain. Biomass feeding did not affect hematological parameters. In the high-dose group, slightly elevated alkaline phosphatase and changes in some urine parameters and an increase in kidney weight in both sexes were noted. Histopathology examinations did not reveal adverse effects except for a marginal increase in pigment in the straight proximal tubule of the kidney in 5/10 female rats treated with the high-dose. These changes were not considered as toxicologically significant. Although the rats in high-dose group received about 9% more fat, it is unlikely that this confounding factor significantly altered the outcome. The no-observed adverse-effect-levels (NOAEL) of the astaxanthin-rich biomass for male and female rats were determined as 14,161 and 17,076mg/kg body weight/day, or 465 and 557mg astaxanthin/kg/day, respectively, the highest dose tested.

Safety assessment of aqueous olive pulp extract as an antioxidant or antimicrobial agent in foods.

The olive fruit, its oil and the leaves of the olive tree have a rich history of nutritional, medicinal and ceremonial uses. Olive oil, table olives and olive products are an important part of the Mediterranean diet, the greatest value of which may be due to olive polyphenols that contribute to the modulation of the oxidative balance in vivo. The objective of this review is to examine the available safety/toxicity literature on olive polyphenols, particularly hydroxytyrosol, to determine the safety-in-use of a standardized aqueous olive pulp extract (HIDROX). Among the polyphenols found in the extract, the major constituent of biological significance is hydroxytyrosol (50-70%). In oral bioavailability studies, urinary excretion of hydroxytyrosol and its glucuronide was found to be associated with the intake of hydroxytyrosol. Oral bioavailability of hydroxytyrosol in olive oil and in an aqueous solution was reported as 99% and 75%, respectively. In comparative studies, urinary excretion of hydroxytyrosol was greater in humans than in rats. The LD(50) of the extract and hydroxytyrosol was reported to be greater than 2000 mg/kg. In a subchronic study, the no observed adverse effect level (NOAEL) of the extract in rats was found to be 2000 mg/kg/day. In developmental and reproductive toxicity studies, HIDROX did not cause toxicity at levels up to 2000 mg/kg/day. In an in vivo micronucleus assay, oral exposure of rats to HIDROX at dose levels up to 5000 mg/kg/day for 29 days did not induce increases in polychromatic erythrocytes in bone marrow. Based on the available studies of the extract and polyphenols, and a history of exposure and use of components of the extract through table olives, olive products and olive oil, the consumption of HIDROX is considered safe at levels up to 20 mg/kg/day.

Safety assessment of esters of p-hydroxybenzoic acid (parabens).

Parabens are widely used as preservatives in food, cosmetic and pharmaceutical products. Acute, subchronic, and chronic studies in rodents indicate that parabens are practically non-toxic. Parabens are rapidly absorbed, metabolized, and excreted. In individuals with normal skin, parabens are, for the most part, non-irritating and non-sensitizing. However, application of compounds containing parabens to damaged or broken skin has resulted in sensitization. Genotoxicity testing of parabens in a variety of in vitro and in vivo studies primarily gave negative results. The paraben structure is not indicative of carcinogenic potential, and experimental studies support these observations. Some animal studies have reported adverse reproductive effects of parabens. In an uterotrophic assay, methyl and butyl paraben administered orally to immature rats were inactive, while subcutaneous administration of butyl paraben produced a weak positive response. The ability of parabens to transactivate the estrogen receptor in vitro increases with alkyl group size. The detection of parabens in a small number of breast tumor tissue samples and adverse reproductive effects of parabens in animals has provoked controversy over the continued use of these substances. However, the possible estrogenic hazard of parabens on the basis of the available studies is equivocal, and fails to consider the metabolism and elimination rates of parabens, which are dose, route, and species dependent. In light of the recent controversy over the estrogenic potential of parabens, conduct of a reproductive toxicity study may be warranted.

Pristane-induced effects on cytochrome P-4501A, ornithine decarboxylase and putrescine in rats.

The effects of pristane (2,6,10,14-tetramethylpentadecane) on cytochrome P-4501A (cP4501A) activity in microsomes, as well as on ornithine decarboxylase (ODC) activity and concomitant putrescine levels were examined in Copenhagen rats. In general, pristane treatment led to increased cP4501A levels when compared to basal levels, while co-treatment with 3-methylcholanthrene (3-MC) and pristane elicited augmented cP4501A responses when compared to responses induced by 3-MC alone. Increases in both ODC activity and putrescine levels were also observed in pristane treated rats. Collectively, these results indicate that pristane influences cP4501A activity and elicits promoter-like responses as reflected in elevated ODC activity and increased amount of putrescine.

Role of tissue repair in toxicologic interactions among hepatotoxic organics.

It is widely recognized that exposure to combinations or mixtures of chemicals may result in highly exaggerated toxicity even though individual chemicals might not be toxic at low doses. Chemical mixtures may also cause additive or less than additive toxicity. From the perspective of public health, highly exaggerated toxicity is of significant concern. Assessment of risk from exposure to chemical mixtures requires knowledge of the underlying mechanisms. Previous studies from this laboratory have shown that nontoxic doses of chlordecone (10 ppm, 15 days) and carbon tetrachloride (CCl4) (100 microliters/kg) interact at the biologic interface, resulting in potentiated liver injury and 67-fold amplification of CCl4 lethality. In contrast, although interaction between phenobarbital and CCl4 leads to even higher injury, animal survival is unaffected because of highly stimulated compensatory tissue repair. A wide variety of additional experimental evidence confirms the central role of stimulated tissue repair as a decisive determinant of the final outcome of liver injury inflicted by hepatotoxicants. These findings led us to propose a two-stage model of toxicity. In this model, tissue injury is inflicted in stage one by the well-described mechanisms of toxicity, whereas in stage two the ultimate toxic outcome is determined by whether timely and sufficient tissue repair response accompanies this injury. In an attempt to validate this model, dose-response relationships for injury and tissue repair as opposing responses have been developed for model hepatotoxicants. Results of these studies suggest that tissue repair increases in a dose-dependent manner, restraining injury up to a threshold dose, whereupon it is inhibited, allowing an unrestrained progression of injury. These findings indicate that tissue repair is a quantifiable response to toxic injury and that inclusion of this response in risk assessment may help in fine-tuning prediction of toxicity outcomes.

Temporal changes in tissue repair permit survival of diet-restricted rats from an acute lethal dose of thioacetamide.

Although, diet restriction (DR) has been shown to substantially increase longevity while reducing or delaying the onset of age-related diseases, little is known about the mechanisms underlying the beneficial effects of DR on acute toxic outcomes. An earlier study (S. K. Ramaiah et al., 1998, Toxicol. Appl. Pharmacol. 150, 12-21) revealed that a 35% DR compared to ad libitum (AL) feeding leads to a substantial increase in liver injury of thioacetamide (TA) at a low dose (50 mg/kg, i.p.). Higher liver injury was accompanied by enhanced survival. A prompt and enhanced tissue repair response in DR rats at the low dose (sixfold higher liver injury) occurred, whereas at equitoxic doses (50 mg/kg in DR and 600 mg/kg in AL rats) tissue repair in AL rats was substantially diminished and delayed. The extent of liver injury did not appear to be closely related to the extent of stimulated tissue repair response. The purpose of the present study was to investigate the time course (0-120 h) of liver injury and liver tissue repair at the high dose (600 mg TA/kg, i.p., lethal in AL rats) in AL and DR rats. Male Sprague-Dawley rats (225-275 g) were 35% diet restricted compared to their AL cohorts for 21 days and on day 22 they received a single dose of TA (600 mg/kg, i.p.). Liver injury was assessed by plasma ALT and by histopathological examination of liver sections. Tissue repair was assessed by [3H]thymidine incorporation into hepatonuclear DNA and proliferating cell nuclear antigen (PCNA) immunohistochemistry during 0-120 h after TA injection. In AL-fed rats hepatic necrosis was evident at 12 h, peaked at 60 h, and persisted thereafter until mortality (3 to 6 days). Peak liver injury was approximately twofold higher in DR rats compared to that seen in AL rats. Hepatic necrosis was evident at 36 h, peaked at 48 h, persisted until 96 h, and returned to normal by 120 h. Light microscopy of liver sections revealed progression of hepatic injury in AL rats whereas injury regressed completely leading to recovery of DR rats by 120 h. Progression of injury led to 90% mortality in AL rats vs 30% mortality in DR group. In the surviving AL rats, S-phase DNA synthesis was evident at 60 h, peaked at 72 h, and declined to base level by 120 h, whereas in DR rats S-phase DNA synthesis was evident at 36 h and was consistently higher until 96 h reaching control levels by 120 h. PCNA studies showed a corresponding increase in cells in S and M phase in the AL and DR groups. DR resulted in abolition of the delay in tissue repair associated with the lethal dose of TA in ad libitum rats. Temporal changes and higher tissue repair response in DR rats (earlier and prolonged) are the conduits that allow a significant number of diet restricted rats to escape lethal consequence.

Tissue repair response as a function of dose during trichloroethylene hepatotoxicity.

Trichloroethylene (TCE), a widely used organic solvent and degreasing agent, is regarded as a hepatotoxicant. The objective of the present studies was to investigate whether the extent and timeliness of tissue repair has a determining influence on the ultimate outcome of hepatotoxicity. Male Sprague-Dawley rats (200-250 g) were injected with a 10-fold dose range of TCE and hepatotoxicity and tissue repair were studied during a time course of 0 to 96 h. Light microscopic changes as evaluated by H&E-stained liver sections revealed a dose-dependent necrosis of hepatic cells. Maximum liver cell necrosis was observed at 48 h after the TCE administration. However, liver injury as assessed by plasma sorbitol dehydrogenase (SDH) showed a dose response over a 10-fold dose range only at 6 h, whereas alanine aminotransferase (ALT) did not show a dose response at any of the time points studied. A low dose of TCE (250 mg/kg) showed an increase in SDH at all time points up to 96 h without peak levels, whereas higher doses showed peak only at 6 h. At later time points SDH declined but remained above normal. In vitro addition of trichloroacetic acid, a metabolite of TCE to plasma, decreased the activities of SDH and ALT indicating that metabolites formed during TCE toxicity may interfere with plasma enzyme activities in vivo. This indicates that the lack of dose-related increase in SDH and ALT activities may be because of interference by the TCE metabolite. Tissue regeneration response as measured by [3H]thymidine incorporation into hepatocellular nuclear DNA was stimulated maximally at 24 h after 500 mg/kg TCE administration. A higher dose of TCE led to a delay and diminishment in [3H]thymidine incorporation. At a low dose of TCE (250 mg/kg) [3H]thymidine incorporation peaked at 48 h and this could be attributed to very low or minimal injury caused by this dose. With higher doses tissue repair was delayed and attenuated allowing for unrestrained progression of liver injury. These results support the concept that the toxicity and repair are opposing responses and that a dose-related increase in tissue repair represents a dynamic, quantifiable compensatory mechanism.

Effect of sodium fluoride on hepatic microsomal mixed-function oxidase system in newborn and adult male rats.

Administration i.p. of sodium fluoride at 2.5 or 5.0 mg/kg induced the mixed-function oxidase system in both newborn and adult rats; at 10.0 mg/kg there was induction in newborn rats and inhibition in adult rats, and at 20.0 mg/kg, there was inhibition in both newborn and adults.

Alterations in drug metabolising enzymes and lipid peroxidation in different rat tissues by fluoride.

Sodium fluoride at a dose level of 5.0 mg/kg enhanced aminopyrine N-demethylase and NADPH cytochrome c reductase activities and cytochrome P450 and cytochrome b5 levels in rat liver, kidney, lung, intestine and testis, whereas acetanilide hydroxylase activity remained unchanged in kidney and lung and was increased in liver, intestine and testis. Sodium fluoride at 20.0 mg/kg caused a decrease in aminopyrine N-demethylase, acetanilide hydroxylase and NADPH cytochrome c reductase activities and cytochrome P450 and cytochrome b5 levels in all tissues, except for an increase in NADPH cytochrome c reductase activity in the intestine and testis. Fluoride at both dose levels produced only marginal changes in glutathione-S-transferase activity except for a 4-fold increase in the testis at 5.0 mg/kg. Sodium fluoride at 5.0 mg/kg increased lipid peroxidation in all tissues studied. At 20.0 mg/kg there was a decrease in lipid peroxidation in liver, lung and testis and an increase in kidney and intestine.

Chronic study of diacylglycerol oil in rats.

The objective of the present study was to evaluate the effects of diacylglycerol oil following long-term administration to rats. Diacylglycerol oil is an edible oil with comparable taste and physicochemical properties of several naturally occurring oils. Diacylglycerol oil can be used as a replacement for any generally used edible oil in the home and has been approved for use in cooking oil in Japan. Male and female Sprague-Dawley rats were divided into four groups and fed low-fat (1.7%) basal diets containing an edible oil composed of rapeseed, corn, high linoleic safflower and high oleic safflower oils at 5.3% (control group 1); an edible oil composed of rapeseed and soybean oils at 5.3% (control group 2); diacylglycerol oil at 2.65% plus edible oil composed of rapeseed, corn, high linoleic safflower and high oleic safflower oils at 2.65% (low-dose group); and diacylglycerol oil at 5.3% (high-dose group) for 2 years. Interim sacrifices were conducted at weeks 30 and 77 and the study was terminated following 105 weeks of feeding. No compound-related effects were noted on clinical signs, body weights, food consumption, cumulative survival rates, hematology, blood chemistry, urinalysis, organ weights or on microscopic non-neoplastic changes. Compared to control group 2, but not control group 1, there was a significant increase in the number of high-dose group females with either benign or malignant epithelial mammary gland neoplasms. These changes were not considered biologically significant, because the tumor incidence was not similar in control group 1 and 2, and the neoplastic findings were not dose related. In summary, the two-year chronic rat study revealed no toxicologically significant or treatment-related effects of diacylglycerol oil consumption at levels of up to 5.3% in the diet.

Safety assessment of propyl paraben: a review of the published literature.

Propyl paraben (CAS no. 94-13-3) is a stable, non-volatile compound used as an antimicrobial preservative in foods, drugs and cosmetics for over 50 years. It is an ester of p-hydroxybenzoate. Propyl paraben is readily absorbed via the gastrointestinal tract and dermis. It is hydrolyzed to p-hydroxybenzoic acid, conjugated and the conjugates are rapidly excreted in the urine. There is no evidence of accumulation. Acute toxicity studies in animals indicate that propyl paraben is relatively non-toxic by both oral and parenteral routes, although it is mildly irritating to the skin. Following chronic administration, no-observed-effect levels (NOEL) as high as 1200-4000 mg/kg have been reported and a no-observed-adverse-effect level (NOAEL) in the rat of 5500 mg/kg is posited. Propyl paraben is not carcinogenic, mutagenic or clastogenic. It is not cytogenic in vitro in the absence of carboxyesterase inhibitors. The mechanism of propyl paraben may be linked to mitochondrial failure dependent on induction of membrane permeability transition accompanied by the mitochondrial depolarization and depletion of cellular ATP through uncoupling of oxidative phosphorylation. Sensitization has occurred when medications containing parabens have been applied to damaged or broken skin. Parabens have been implicated in numerous cases of contact sensitivity associated with cutaneous exposure, but high concentrations of 5-15% in patch testing are needed to elicit reaction in susceptible individuals. Allergic reactions to ingested parabens have been reported, although rigorous evidence of the allergenicity of ingested paraben is lacking.

Evaluation of the health aspects of methyl paraben: a review of the published literature.

Methyl paraben (CAS No. 99-76-3) is a methyl ester of p-hydroxybenzoic acid. It is a stable, non-volatile compound used as an antimicrobial preservative in foods, drugs and cosmetics for over 50 years. Methyl paraben is readily and completely absorbed through the skin and from the gastrointestinal tract. It is hydrolyzed to p-hydroxybenzoic acid, conjugated, and the conjugates are rapidly excreted in the urine. There is no evidence of accumulation. Acute toxicity studies in animals indicate that methyl paraben is practically non-toxic by both oral and parenteral routes. In a population with normal skin, methyl paraben is practically non-irritating and non-sensitizing. In chronic administration studies, no-observed-effect levels (NOEL) as high as 1050 mg/kg have been reported and a no-observed-adverse-effect level (NOAEL) in the rat of 5700 mg/kg is posited. Methyl paraben is not carcinogenic or mutagenic. It is not teratogenic or embryotoxic and is negative in the uterotrophic assay. The mechanism of cytotoxic action of parabens may be linked to mitochondrial failure dependent on induction of membrane permeability transition accompanied by the mitochondrial depolarization and depletion of cellular ATP through uncoupling of oxidative phosphorylation. Parabens are reported to cause contact dermatitis reactions in some individuals on cutaneous exposure. Parabens have been implicated in numerous cases of contact sensitivity associated with cutaneous exposure; however, the mechanism of this sensitivity is unknown. Sensitization has occurred when medications containing parabens have been applied to damaged or broken skin. Allergic reactions to ingested parabens have been reported, although rigorous evidence of the allergenicity of ingested paraben is lacking.

Safety assessment of (-)-hydroxycitric acid and Super CitriMax, a novel calcium/potassium salt.

(-)-Hydroxycitric acid (HCA) is a principle constituent (10-30%) of the dried fruit rind of Garcinia cambogia, a plant native to Southeastern Asia. The dried rind has been used for centuries throughout Southeast Asia as a food preservative, flavoring agent and carminative. Extensive experimental studies show that HCA inhibits fat synthesis and reduces food intake. The objective of this review is to systematically review the available safety/toxicity literature on HCA to determine its safety in-use. The primary mechanism of action of HCA appears to be related to its ability to act as a competitive inhibitor of the enzyme ATP-citrate lyase, which catalyzes the conversion of citrate and coenzyme A to oxaloacetate and acetyl coenzyme A (acetyl-CoA), primary building blocks of fatty acid and cholesterol synthesis. Super CitriMax, a novel calcium/potassium-HCA extract (HCA-SX), is considerably more soluble and bioavailable than calcium-based HCA ingredients. Acute oral toxicity studies in animals demonstrate that CitriMax (50% HCA as calcium salt) has a low acute oral toxicity. In a subchronic study in rats, the gavage administration of HCA-SX at doses up to 2500 mg/kg/day for a period of 90 days caused a significant decrease in body weight and reduction in feed consumption without any adverse effects. The structure, mechanism of action, long history of use of HCA and other toxicity studies indicate that HCA-SX is unlikely to cause reproductive or developmental effects. HCA-SX was not mutagenic in the presence or absence of metabolic activation in Ames genotoxicity assays in strains TA98 and TA102. HCA-SX-induced increases in number of revertants in other strains (TA100 and TA1535 in the absence of metabolic activation and in strain TA1537 in the presence of metabolic activation) but these were not considered as biologically indicative of a mutagenic effect. In several, placebo-controlled, double-blind trials employing up to 2800 mg/day HCA, no treatment-related adverse effects were reported. There is sufficient qualitative and quantitative scientific evidence, including animal and human data suggesting that intake of HCA at levels up to 2800 mg/day is safe for human consumption.

Hepatic failure leads to lethality of chlordecone-amplified hepatotoxicity of carbon tetrachloride.

Chlordecone (Kepone) amplification of CCl4 toxicity occurs at small, nontoxic levels of chlordecone and CCl4 and results in highly increased irreversible hepatotoxicity culminating in lethality. Although it is generally assumed that CCl4 lethality is due to hepatic failure, no definitive studies are available in the literature bridging massive liver failure and death. The present studies were designed to evaluate whether hepatic failure is the cause of the lethality during chlordecone-amplified CCl4 toxicity. Male Sprague-Dawley rats were maintained on control or a chlordecone (10 ppm) diet for 15 days and injected with CCl4 (100 microliters/kg, ip) on Day 16. Rats were killed at 0, 6, 12, 24, 36, and 48 hr after CCl4 challenge. Hepatic failure was evaluated by measuring plasma glucose, ammonia, bilirubin, aspartate transaminase (AST), alanine transaminase (ALT), sorbitol dehydrogenase (SDH), hepatic ATP, glycogen, and by histological and histomorphometric analyses. Plasma creatinine, urea, and kidney histopathology were also assessed for possible renal injury. As expected CCl4 administration to chlordecone-pretreated rats resulted in 20% lethality by 36 hr, which progressed with time, and all rats died within 72 hr. A significant and progressive hypoglycemia was observed with a 60% reduction in plasma glucose at 48 hr. Hepatic glycogen content dropped precipitously. Similarly, hepatic ATP levels remained suppressed (80% of control) at all the time points studied. Plasma ammonia levels were significantly elevated, and by 48 hr, a threefold increase was observed. Plasma ALT, AST, SDH, and bilirubin increased progressively until the death of rats receiving the chlordecone + CCl4 combination.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine triphosphate protection of chlordecone-amplified CCl4 hepatotoxicity and lethality.

Dietary exposure to a nontoxic level of chlordecone (10 ppm for 15 days) followed by a single exposure to a subtoxic dose of CCl4 (100 microliters/kg, ip) is known to result in a 67-fold amplification of CCl4 toxicity. The hypothesis that the underlying mechanism is due to incapacitation of hepatocytes leading to an ablation of the early-phase hormetic response of tissue repair as a consequence of precipitous decline in hepatic glycogen and ATP, received experimental support from Mehendale in 1990. The present study was designed to investigate if direct administration of ATP to rats maintained on the chlordecone diet would result in protection from the hepatotoxic and lethal effects of the chlordecone+CCl4 combination. Male Sprague-Dawley rats (125-150 g) were maintained either on a diet containing no added contaminants (control) or on a diet containing 10 ppm chlordecone for 15 days, and were challenged with CCl4 (100 microliters/kg, ip) on day 16. Without ATP administration all rats died within 72 h, while administration of ATP (100 mg/rat, sc) to chlordecone-pretreated rats at -1, +1, 3, 5, 12, 24 and 36 h of CCl4 injection resulted in 100% survival. Injection of ATP, at -1, +1, 3 and 5 h of CCl4 administration to chlordecone pretreated rats decreased plasma enzyme elevations (alanine and aspartate aminotransferase, sorbitol dehydrogenase) as well as substantially preventing elevation of plasma bilirubin levels at 6, 12 and 24 h. Hepatic ATP levels were also elevated at 6 and 12 h, but not at 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Protection from chlordecone-amplified carbon tetrachloride toxicity by cyanidanol: biochemical and histological studies.

Chlordecone (CD) pretreatment is well known to greatly potentiate CCl4 toxicity. Previous work has shown that suppression of hepatocellular regeneration permits an ordinarily limited liver injury to progress in an irreversible manner. Insufficient hepatocellular energy has been proposed as a mechanism for suppressed hepatocellular regeneration. Since cyanidanol reportedly increases cellular ATP, this compound was employed to test the above hypothesis. The present study was designed to investigate the sequential biochemical and histological changes over a time course of 120 hr after CCl4 administration. Male Sprague-Dawley rats (125-150 g) were maintained on 10 ppm CD diet for 15 days and were challenged with either a standard protocol dose (100 microliters/kg) or a low (50 microliters/kg, L) dose of CCl4. Cyanidanol pretreatment at 48, 24, and 2 hr before CCl4 administration to rats maintained on CD diet resulted in 100 or 70% animal survival, for CCl4 (L) or the standard dose of CCl4, respectively. Preliminary studies indicated that neither simultaneous nor subsequent administration of cyanidanol with CCl4 challenge affords such protection. Prior treatment with cyanidanol and a latency period were found necessary for protection. Without cyanidanol, CD + CCl4 combination caused 50 and 100% lethality after CCl4 (L) and the standard dose, respectively, while the same doses of CCl4 alone did not cause lethal effects. Plasma enzymes (alanine aminotransferase, aspartate aminotransferase, sorbitol dehydrogenase) in control rats showed only moderate and transient increases after CCl4 challenge. The combination of CD + standard dose of CCl4 resulted in progressive and marked elevations of all three serum enzymes at all time intervals until the death of animals. Cyanidanol pretreatment resulted in significant decline in the plasma enzyme elevations at later time points. Cyanidanol pretreatment increased hepatic ATP synthesis in control or CD rats. CCl4 administration to control rats did not alter hepatic ATP levels, while in CD-fed rats hepatic ATP levels were significantly decreased. Cyanidanol pretreatment to CD + CCl4 combination-treated rats did not significantly prevent the decline in hepatic ATP and glycogen levels. However, in the surviving rats a recovery in these parameters was observed. Light microscopic examination of livers from animals that received CCl4 alone revealed only marginal cellular injury, at early time points only. However, CCl4 challenge to rats maintained on CD resulted in progressive injury, characterized by the appearance of ballooned cells, necrotic cells, and cells with lipid droplets in the liver. Cyanidanol pretreatment to these rats caused decreased vacuolation and significantly reduced the progression of liver necrosis.(ABSTRACT TRUNCATED AT 400 WORDS)

Protection from chlordecone-amplified carbon tetrachloride toxicity by cyanidanol: regeneration studies.

Previous work has shown that chlordecone (CD)-amplified CCl4 hepatotoxicity and lethality can be mitigated by pretreatment with cyanidanol. These studies also revealed that stimulated hepatocellular regeneration might play an important role in the cyanidanol protection of CD-amplified CCl4 toxicity. The present studies conducted over a time course of 0 to 120 hr after CCl4 challenge describe sequential changes in hepatic [3H]thymidine incorporation into hepatocellular nuclear DNA, polyamines and related enzymes, and histomorphometry of liver sections from variously treated rats. Male Sprague-Dawley rats (125-150 g) were maintained on a control diet or on a diet contaminated with CD (10 ppm) for 15 days and/or pretreated with cyanidanol (250 mg/kg, ip) at 48, 24, and 2 hr before a single ip injection of either a standard protocol dose (100 microliters/kg) or a low dose (50 microliters/kg, L) of CCl4 on Day 16 of the dietary protocol. Cyanidanol pretreatment significantly stimulated the hepatic [3H]thymidine incorporation into hepatocellular nuclear DNA of control rats irrespective of CD pretreatment. Similarly, polyamine metabolism was altered favorably for cell division, although mitotic index (metaphase) was not increased. Cyanidanol-stimulated [3H]thymidine incorporation was highly suppressed in rats receiving the CD + CCl4 standard dose combination treatment up to 36 hr, but after this time point a marked increase was observed. Hepatocellular regeneration, quantified histomorphometrically as volume density of cells in metaphase, was progressively increased in rats protected from CD + CCl4 interaction by cyanidanol, starting at 36 hr and lasting until 72 hr. Favorably altered polyamine metabolism was evident from the stimulated ornithine decarboxylase, as well as from the stimulated interconversion of the higher polyamines to maintain increased concentration of putrescine. Challenge by the same dose of CCl4 (100 microliters/kg) to CD-pretreated rats not protected by cyanidanol failed to cause any increase in [3H]thymidine incorporation up to 36 hr and resulted in animal death starting at 36 hr. In the surviving rats, [3H]thymidine incorporation at 48 hr was increased, but was less than 50% of the increase observed in the cyanidanol group. In these rats, attenuation in the stimulation of cell division and insufficiently increased putrescine levels were observed, which are consistent with the inadequate level of hepatocellular regeneration. With rats receiving CD + CCl4(L) combination, the [3H]thymidine incorporation at 48 hr was less than 50% of the increase of cyanidanol-protected rats. Cyanidanol pretreatment to the CD + CCl4 group of rats prevented the decrease in the hepatic DNA levels.(ABSTRACT TRUNCATED AT 400 WORDS)