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 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 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.

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.

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 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.

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 evaluation of dietary aluminum.

Aluminum is a nonessential metal to which humans are frequently exposed. Aluminum in the food supply comes from natural sources, water used in food preparation, food ingredients, and utensils used during food preparations. The amount of aluminum in the diet is small, compared with the amount of aluminum in antacids and some buffered analgesics. The healthy human body has effective barriers (skin, lungs, gastrointestinal tract) to reduce the systemic absorption of aluminum ingested from water, foods, drugs, and air. The small amount of aluminum (<1%) that is systemically absorbed is excreted principally in the urine and, to a lesser extent, in the feces. No reports of dietary aluminum toxicity to healthy individuals exist in the literature. Aluminum can be neurotoxic, when injected directly into the brains of animals and when accidentally introduced into human brains (by dialysis or shrapnel). A study from Canada reports cognitive and other neurological deficits among groups of workers occupationally exposed to dust containing high levels of aluminum. While the precise pathogenic role of aluminum in Alzheimer's disease (AD) remains to be defined, present data do not support a causative role for aluminum in AD. High intake of aluminum from antacid for gastrointestinal ailments has not been reported to cause any adverse effects and has not been correlated with neurotoxicity or AD. Foods and food ingredients are generally the major dietary sources of aluminum in the United States. Cooking in aluminum utensils often results in statistically significant, but relatively small, increases in aluminum content of food. Common aluminum-containing food ingredients are used mainly as preservatives, coloring agents, leavening agents, anticaking agents, etc. Safety evaluation and approval of these ingredients by the Food and Drug Administration indicate that these aluminum-containing compounds are safe for use in foods.

Evaluation of health aspects of kojic acid in food.

Kojic acid is a fungal metabolite commonly produced by many species of Aspergillus, Acetobacter, and Penicillium. The Aspergillus flavus group has traditionally been used in the production of a number of foods, including miso (soybean paste), shoyu (soy sauce), and sake. Kojic acid is widely used as a food additive for preventing enzymatic browning, and in cosmetic preparations as a skin-lightening or bleaching agent. Because kojic acid is often produced during the fermentation of historically used dietary staples, it has a long history of consumption. Various types of compounds, such as glucose, sucrose, acetate, ethanol, arabinose, and xylose, have been used as carbon sources for kojic acid production. Different Aspergillus species are known to produce variable amounts of kojic acid. The mechanism of action of kojic acid is well defined and it has been shown to act as a competitive and reversible inhibitor of animal and plant polyphenol oxidases, xanthine oxidase, and D- and some L-amino acid oxidases. The structure of kojic acid indicates a relatively simple route of metabolism much like dietary hexoses. Acute or subchronic toxicity resulting from an oral dose has not been reported, but convulsions may occur if kojic acid is injected. Results of mutagenicity studies are mixed, but in the in vivo mammalian dominant lethal assay, kojic acid was proven negative. Continuous administration of high doses of kojic acid in mice resulted in induction of thyroid adenomas in both sexes. Kojic acid reversibly affects thyroid function primarily by inhibiting iodine uptake, leading to decreases in T3 and T4 and increase in TSH. Increased TSH from pituitary gland in turn stimulates thyroid hyperplasia. Several lines of evidence indicate that the proliferative effects of kojic acid on thyroid are not related to a genotoxic pathway. The risk of functional inhibition of iodine uptake and its metabolism (organification) and thyroid tumor induction by kojic acid in humans appears to be extremely low. Based on the literature reviewed and discussed here, consumption of kojic acid at levels normally found in food does not present a concern for safety.

Enhanced hepatotoxicity and toxic outcome of thioacetamide in streptozotocin-induced diabetic rats.

Diabetes is known to potentiate thioacetamide (TA)-induced liver injury via enhanced bioactivation. Little attention has been given to the role of compensatory tissue repair on ultimate outcome of hepatic injury in diabetes. The objective of this study was to investigate the effect of diabetes on TA-induced liver injury and lethality and to investigate the underlying mechanisms. We hypothesized that hepatotoxicity of TA in diabetic rats would increase due to enhanced bioactivation-mediated liver injury and also due to compromised compensatory tissue repair, consequently making a nonlethal dose of TA lethal. On day 0, male Sprague-Dawley rats (250-300 g) were injected with streptozotocin (STZ, 60 mg/kg ip) to induce diabetes. On day 10 the STZ-induced diabetic rats and the nondiabetic rats received a single dose of TA (300 mg/kg ip). This normally nonlethal dose of TA caused 90% mortality in the STZ-induced diabetic rats. At various times (0-60 h) after TA administration, liver injury was assessed by plasma alanine aminotransferase (ALT), sorbitol dehydrogenase (SDH), and liver histopathology. Liver function was evaluated by plasma bilirubin. Cell proliferation and tissue repair were evaluated by [(3)H]thymidine ((3)H-T) incorporation and proliferating cell nuclear antigen (PCNA) assays. In the nondiabetic rat, liver necrosis peaked at 24 h and declined thereafter toward normal by 60 h. In the STZ-induced diabetic rat, however, liver necrosis was significantly increased from 12 h onward and progressed, culminating in liver failure and death. Liver tissue repair studies showed that, in the liver of nondiabetic rats, S-phase DNA synthesis was increased at 36 h and peaked at 48 h following TA administration. However, DNA synthesis was approximately 50% inhibited in the liver of diabetic rats. PCNA study showed a corresponding decrease of cell-cycle progression, indicating that the compensatory tissue repair was sluggish in the diabetic rats. Further investigation of tissue repair by employing equitoxic doses (300 mg TA/kg in the non-diabetic rats; 30 mg TA/kg in the diabetic rats) revealed that, despite equal injury up to 24 h following injection, the tissue repair response in the diabetic rats was much delayed. The compromised tissue repair prolonged liver injury in the diabetic rats. These studies suggest that the increased TA hepatotoxicity in the diabetic rat is due to combined effects of increased bioactivation-mediated liver injury of TA and compromised compensatory tissue repair.

Toxicant-inflicted injury and stimulated tissue repair are opposing toxicodynamic forces in predictive toxicology.

These studies were designed to investigate the dose response for liver injury and tissue repair induced by exposure to four structurally and mechanistically dissimilar hepatotoxicants, individually and as mixtures. The objective was to illuminate the impact of the extent and timeliness of tissue repair on the ultimate outcome of toxicity. Dose-response relationships for trichloroethylene (TCE), allyl alcohol (AA), thioacetamide (TA), and chloroform alone or as mixtures were studied. Male Sprague-Dawley rats (200-250 g) received a single intraperitoneal injection of individual toxicants as well as mixtures of these toxicants. Liver injury was monitored by plasma enzyme (ALT and SDH) levels and histopathology. Tissue regeneration was measured by [3H]thymidine incorporation into hepatic nuclear DNA. Individually, TCE, TA, and AA administration, over a 10- to 12-fold dose range, revealed a dose-related increase in injury as well as tissue repair up to a threshold dose. Beyond this threshold, tissue repair was delayed and attenuated, and liver injury progressed. Mixtures of the four chemicals at the higher doses used in individual dose-response studies resulted in 100% mortality. Hence, mixtures at the lower two doses were selected for further study. Additional lower doses were also included to better understand the dose-response relationship of mixtures. Results of these studies support the observations of individual chemicals. Higher and sustained repair was observed at low dose levels. These studies show that the extent of injury at early time points correlates well with the maximal stimulation of the opposing response of tissue repair. It appears that the toxicity of the mixture employed in these studies is roughly additive and correlates well with tissue repair response. These initial studies suggest that a biologically based mathematical model can be constructed and tested to extrapolate the outcome of toxicity from a given dose of individual compounds as well as their mixtures, where the responses measured are injury on the one hand and compensatory tissue repair on the other.

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.

Diet restriction enhances compensatory liver tissue repair and survival following administration of lethal dose of thioacetamide.

Diet restriction is known to prevent a plethora of age-associated diseases including cancer. However, the effects of diet restriction on noncancer end points are not known. The objective of this study was to investigate whether diet restriction protects against hepatotoxicity of thioacetamide (TA), and if so, to investigate the underlying mechanism. Male Sprague-Dawley rats (250-275 g) were maintained on 65% of their ad libitum (AL) food consumption for a period of 3 weeks and then treated with a single low dose of 50 mg TA/kg i.p.. Plasma enzymes (ALT and SDH), hepatic glycogen levels, and 3H-thymidine incorporation into hepatocellular nuclear DNA were measured during a time course (0-120 h) after TA administration. Liver sections were examined for histopathology, and cell-cycle progression was assessed by proliferating cell nuclear antigen (PCNA) immunohistochemistry. In AL rats hepatic necrosis was evident at 12 h, peaked at 36 h, persisted up to 72 h, and was resolved by 96 h. In the diet-restricted (DR) group hepatic necrosis was observed at 12 h, peaked at 24 h, persisted till 72 h, and was resolved by 96 h. Maximal injury indicated by enzyme elevation occurred in DR rats and was approximately sixfold greater than that observed in the AL group. Histopathological examination of the liver sections revealed liver injury concordant with plasma enzyme elevations. There was a higher and sustained S-phase synthesis in the DR rats compared to AL group. S-phase stimulation was evident at 36 h, peaked at 48 h, and persisted until 96 h in the DR rats, whereas in the AL rats peak S-phase stimulation occurred at 36 h and subsided by 72 h. PCNA studies revealed a corresponding stimulation of cell-cycle progression indicating highly stimulated compensatory tissue repair. The 14-day lethality experiments (600 mg TA/kg i.p.) indicated 70% survival in the DR rats compared to 10% survival in the AL group. Although diet restriction increases hepatotoxic injury of TA, it protects from the lethal outcome by enhanced liver tissue repair. Comparison of liver injury and tissue repair employing an equitoxic dose (600 mg TA/kg in AL rats yields similar liver injury as observed with 50 mg TA/kg in DR rats) revealed that in spite of near equal injury up to 36 h, tissue repair response in DR rats is much higher. The compensatory tissue repair allows the DR rats to escape death in contrast to much lower compensation in AL rats leading to progression of liver injury culminating in death.

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.

Human leukocyte glutathione S-transferase isozyme (class mu) and susceptibility to smoking-related cancers.

Glutathione S-transferase isozyme class mu from human leukocytes has been shown to be dominantly inherited and can be determined by activity measurement directed toward the substrate trans-stilbene oxide. The activity distribution of leukocyte glutathione S-transferase class mu was determined from control healthy nonsmokers, smokers, and smoking-related cancer patients. In a control healthy nonsmoker population, 54% (n = 50) of the subjects showed high levels of glutathione S-transferase class mu activity. In patients with cancers known to be related to smoking, 46% (n = 50) showed higher levels of glutathione S-transferase class mu. Noncancer smokers matched for age and smoking history with cancer patients showed an increased likelihood of having glutathione S-transferase (GST) class mu activity (76%). These results suggest that GST mu may be a cancer susceptibility marker in the case of smokers. In rats, benzo[a]pyrene (1 mg/kg, ip) administration daily for 3 d produced a significant increase in liver glutathione S-transferase class mu. Although these induction studies in experimental animals may not be relevant to humans, there is a possibility that, as in rats, this enzyme may be inducible in humans by polycyclic aromatic hydrocarbons.

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.

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)

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)