Hericium erinaceus Inhibits TNF-α-Induced Angiogenesis and ROS Generation through Suppression of MMP-9/NF-κB Signaling and Activation of Nrf2-Mediated Antioxidant Genes in Human EA.hy926 Endothelial Cells.

Hericium erinaceus (HE) is an edible mushroom that has been shown to exhibit anticancer and anti-inflammatory activities. We investigated the antiangiogenic and antioxidant potentials of ethanol extracts of HE in human endothelial (EA.hy926) cells upon tumor necrosis factor-α- (TNF-α-) stimulation (10 ng/mL). The underlying molecular mechanisms behind the pharmacological efficacies were elucidated. We found that noncytotoxic concentrations of HE (50-200 µg/mL) significantly inhibited TNF-α-induced migration/invasion and capillary-like tube formation of endothelial cells. HE treatment suppressed TNF-α-induced activity and/or overexpression of matrix metalloproteinase-9 (MMP-9) and intercellular adhesion molecule-1 (ICAM-1). Furthermore, HE downregulated TNF-α-induced nuclear translocation and transcriptional activation of nuclear factor-κB (NF-κB) followed by suppression of I-κB (inhibitor-κB) degradation. Data from fluorescence microscopy illustrated that increased intracellular ROS production upon TNF-α-stimulation was remarkably inhibited by HE pretreatment in a dose-dependent manner. Notably, HE triggered antioxidant gene expressions of heme oxygenase-1 (HO-1), γ-glutamylcysteine synthetase (γ-GCLC), and glutathione levels, which may contribute to inhibition of ROS. Increased antioxidant status was associated with upregulated nuclear translocation and transcriptional activation of NF-E2 related factor-2 (Nrf2) in HE treated cells. Our findings conclude that antiangiogenic and anti-inflammatory activities of H. erinaceus may contribute to its anticancer property through modulation of MMP-9/NF-κB and Nrf2-antioxidant signaling pathways.

Antrodia camphorata induces G(1) cell-cycle arrest in human premyelocytic leukemia (HL-60) cells and suppresses tumor growth in athymic nude mice.

Antrodia camphorata is a well-known medicinal mushroom in Taiwan. The broth from a fermented culture of Antrodia camphorata (AC) has been shown to induce apoptosis in cultured human premyelocytic leukemia (HL-60) cells. In the present study, we examined the effects of AC on cell cycle arrest in vitro in HL-60 cells and on tumor regression in vivo using an athymic nude mouse model. We found that AC (20-80 µg mL(-1)) treatment significantly induced G1 cell-cycle arrest in HL-60 cells by reducing the levels of cyclin D1, CDK4, cyclin E, CDK2, cyclin A, and phosphorylation of retinoblastoma protein (p-Rb). Moreover, AC treatment led to significantly increased protein expression levels of CDK inhibitors, including p21(WAF1) and p15(NIK4B). Additionally, AC treatment markedly induced intracellular ROS generation and mitochondrial dysfunction in HL-60 cells. Furthermore, the in vivo study results revealed that AC treatment was effective in terms of delaying the tumor incidence in nude mice that had been inoculated with HL-60 cells as well as in reducing the tumor burden. Histological analysis confirmed that AC treatment significantly modulated the xenografted tumor progression as demonstrated by a reduction in mitotic cells. Our data strongly suggest that Antrodia camphorata could be an anti-cancer agent for human leukemia.

Antrodia salmonea inhibits TNF-α-induced angiogenesis and atherogenesis in human endothelial cells through the down-regulation of NF-κB and up-regulation of Nrf2 signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Antrodia salmonea (AS) is known as a traditional Chinese medicine, but very few biological activities have been reported. MATERIALS AND METHODS: The present study was aimed to investigate the anti-angiogenic and anti-atherosclerotic potential of the fermented culture broth of AS against tumor necrosis factor-α (TNF-α)-stimulated human endothelial (EA.hy 926) cells. RESULTS: The non-cytotoxic concentrations of AS significantly inhibited TNF-α-induced migration/invasion and capillary-like tube formation in EA.hy 926 cells. Furthermore, AS suppressed TNF-α-induced activity and expression of matrix metalloproteinase-9 (MMP-9), and cell-surface expression of intercellular adhesion molecule-1 (ICAM-1), which was associated with abridged adhesion of U937 leukocytes to endothelial cells. Moreover, AS significantly down-regulated TNF-α-induced nuclear translocation and transcriptional activation of nuclear factor κB (NF-κB) followed by suppression of I-κB degradation and phosphorylation of I-κB kinase-α (IKKα). Notably, the protective effect of AS was directly correlated with the increased expression of hemeoxygenase-1 (HO-1) and γ-glutamylcysteine synthetase (γ-GCLC), which was reasoned by nuclear translocation and transactivation of NF-E2 related factor-2 (Nrf2)/antioxidant response element (ARE). Furthermore, HO-1 knockdown by HO-1-specific shRNA diminished the protective effects of AS on TNF-α-stimulated invasion, tube formation, and U937 adhesion in EA.hy 926 cells. CONCLUSIONS: Taken together, these results suggest that Antrodia salmonea may be useful for the prevention of angiogenesis and atherosclerosis.

Gambogenic acid induced mitochondrial-dependent apoptosis and referred to phospho-Erk1/2 and phospho-p38 MAPK in human hepatoma HepG2 cells.

Gambogenic acid, identified from Gamboge, is responsible for anti-tumor effects, and has been shown to be a potential molecule against human cancers. In this study, the molecular mechanism of gambogenic acid-induced apoptosis in HepG2 cells was investigated. Gambogenic acid significantly inhibited cell proliferation and induced apoptosis. Acridine orange/ethidium bromide (AO/EB) staining was used to observe apoptosis, and then confirmed by transmission electron microscopy. Gambogenic acid induced apoptosis and morphological changes in mitochondria, and intracellular reactive oxygen species (ROS) and mitochondrial membrane permeabilization (MMP) in mitochondrial apoptosis pathway were also examined. Results showed that the levels of phospho-p38 and its downstream phospho-Erk1/2 of HepG2 cells increased in time- and concentration-dependent manners after gambogenic acid treatments. Additionally, gambogenic acid increased expression ratio of Bcl-2/Bax in mRNA levels, Western blotting analysis also further confirmed the reduced level of Bcl-2 and increase the expression level of Bax in HepG2 cells. These results indicated that gambogenic acid induced mitochondrial oxidative stress and activated caspases through a caspase-3 and caspase-9-dependent apoptosis pathway. Moreover, gambogenic acid mediated apoptosis and was involved in the phospho-Erk1/2 and phospho-p38 MAPK proteins expression changes in HepG2 cells.

Butein up-regulates the expression of the π class of glutathione S-transferase in rat primary hepatocytes through the ERK/AP-1 pathway.

Induction of phase II enzymes is an important mechanism of chemoprevention. Here we compared the effects of chalcones on the expression of the π class of glutathione S-transferase (GSTP) in rat primary hepatocytes. Hepatocytes were treated with 10 or 25 µM of phloretin or butein for 24 h. Both butein and phloretin dose-dependently increased GSTP protein expression, and the induction potency of butein was stronger than that of phloretin. The increase in GSTP mRNA in cells treated with 25 µM of phloretin and butein was 107% and 211%, respectively (P < 0.05). Butein increased GST enzyme activity by 27% compared with that in the control cells (P < 0.05). In contrast, phloretin had no significant effect on GST enzyme activity. The pTA-luciferase reporter construct with the rat -2.7 kb GSTP promoter region was transiently transfected into Clone 9 liver cells, and the luciferase activity in butein-treated cells was 1.1-fold higher than that in control cells (P < 0.05). GSTP enhancer 1 (GPE1) deletion abolished the induction of reporter activity by butein. The phosphorylation of extracellular signal-regulated kinase (ERK), but not of c-Jun NH2-terminal kinase (JNK) and p38, was stimulated in the presence of butein. Pretreatment with PD98059, an ERK inhibitor, alleviated the increase in activator protein-1 (AP-1)-DNA binding activity and also the activation of GSTP protein expression by butein. Moreover, c-Jun is likely to bind to the GPE1. Silencing of ERK2 by siRNA gene knockdown reduced the butein-induced expression of GSTP. In conclusion, the increased GSTP expression by butein is likely related to the ERK-AP-1 pathway.

Induction of heme oxygenase 1 and inhibition of tumor necrosis factor alpha-induced intercellular adhesion molecule expression by andrographolide in EA.hy926 cells.

Andrographolide is the most abundant diterpene lactone in Andrographis paniculata, which is widely used as a traditional medicine in Southeast Asia. Heme oxygenase 1 (HO-1) is an antioxidant enzyme encoded by a stress-responsive gene. HO-1 has been reported to inhibit the expression of adhesion molecules in vascular endothelial cells (EC). Intercellular adhesion molecule (ICAM-1) is an inflammatory biomarker that is involved in the adhesion of monocytes to EC. In this study, we investigated the effect of andrographolide on the expression of ICAM-1 induced by tumor necrosis factor alpha (TNF-alpha) in EA.hy926 cells and the possible mechanisms involved. Andrographolide (2.5-7.5 microM) inhibited the TNF-alpha-induced expression of ICAM-1 in a dose-dependent manner and resulted in a decrease in HL-60 cell adhesion to EA.hy926 cells (p < 0.05). In parallel, andrographolide significantly induced the expression of HO-1 in a concentration-dependent fashion (p < 0.05). Andrographolide increased the rate of nuclear translocation of nuclear factor erythroid 2-related 2 (Nrf2) and induced antioxidant response element-luciferase reporter activity. Transfection with HO-1-specific small interfering RNA knocked down HO-1 expression, and the inhibition of expression of ICAM-1 by andrographolide was significantly reversed. These results suggest that stimulation of Nrf2-dependent HO-1 expression is involved in the suppression of TNF-alpha-induced ICAM-1 expression exerted by andrographolide.

Soy protein with and without isoflavones fails to substantially increase postprandial antioxidant capacity.

Five methods for the assessment of antioxidant capacity [whole plasma conjugated diene formation, low-density lipoprotein oxidation susceptibility, ferric-reducing ability of plasma, oxygen radical absorbance capacity and perchloric-acid-treated oxygen radical absorbance capacity (PCA-ORAC)] were used in a randomized, double blind, crossover study to determine the acute postprandial antioxidant protection imparted by the isoflavone component of soy. On separate days, 16 subjects consumed one of three isocaloric shakes containing 25 g of protein in the form of soy, with 107 mg of total aglycone units of isoflavones, soy with trace isoflavones (<4 mg) or total milk protein. Blood was collected at baseline, 4 h, 6 h and 8 h after consumption. Antioxidant capacity, serum isoflavone levels, fat-soluble antioxidants and plasma vitamin C levels were evaluated. Repeated measures analysis of variance showed no significant differences (P=.05) within treatments over time in four of five antioxidant capacity measurements. Significant differences over time between the soy with trace isoflavones and the total milk protein group were observed using the PCA-ORAC assay. It can be concluded that, on an acute basis, a significant increase in serum antioxidant capacity is not detectable following consumption of soy protein.

Metabolism of terephthalic acid and its effects on CYP4B1 induction.

OBJECTIVE: To investgate the metabolism of terephthalic acid (TPA) in rats and its mechanism. Methods Metabolism was evaluated by incubating sodium terephthalate (NaTPA) with rat normal liver microsomes, or with microsomes pretreated by phenobarbital sodium, or with 3-methycholanthrene, or with diet control following a NADPH-generating system. The determination was performed by high performance liquid chromatography (HPLC), and the mutagenic activation was analyzed by umu tester strain Salmonella typhimurium NM2009. Expression of CYP4B1 mRNA was detected by RT-PCR. Results The amount of NaTPA (12.5-200 micromol x L(-1)) detected by HPLC did not decrease in microsomes induced by NADPH-generating system. Incubation of TPA (0.025-0.1 mmol x L(-1)) with induced or noninduced liver microsomes in an NM2009 umu response system did not show any mutagenic activation. TPA exposure increased the expression of CYP4B 1 mRNA in rat liver, kidney, and bladder. CONCLUSION: Lack of metabolism of TPA in liver and negative genotoxic data from NM2009 study are consistent with other previous short-term tests, suggesting that the carcinogenesis in TPA feeding animals is not directly interfered with TPA itself and/or its metabolites.

Fenvalerate-induced alterations in calcium homeostasis in rat ovary.

OBJECTIVE: To observe the effects of fenvalerate on calcium homeostasis in rat ovary. METHODS: Female Sprague-Dawley rats were orally given fenvalerate at daily doses of 0.00, 1.91, 9.55, and 31.80 mg/kg for four weeks. The ovary ultrastucture was observed by electron microscopy. Serum free calcium concentration was measured by atomic absorption spectrophotometry. The activities of phosphorylase a in rat ovary were evaluated by the chromatometry. The total content of calmodulin in ovary was estimated by ELISA at each stage of estrous cycle. Radioimmunoassay (RIA) was used to evaluate the level of serum progesterone. RESULTS: Histopathologically, damages of ovarian corpus luteum cells were observed. An increase in serum free calcium concentration was observed in rats treated with 31.80 mg/kg fenvalerate. The activities of phosphorylase a enhanced in all treated groups, and fenvalerate increased the total content of calmodulin significantly in estrus period. Serum progesterone levels declined in fenvalerate exposed rats in diestrus. CONCLUSION: Fenvalerate interferes with calcium homeostasis in rat ovary. Also, the inhibitory effects of fenvalerate on serum progesterone levels may be mediated partly through calcium signals.

Modification of N-Methyl-N-Nitrosourea initiated bladder carcinogenesis in Wistar rats by terephthalic acid.

The effect of terephthalic acid (TPA) on urinary bladder carcinogenesis was examined. Male Wistar rats were initiated by injection of N-Methyl-N-Nitrosourea (MNU) (20 mg/kg b.w. ip) twice a week for 4 weeks, then given basal diet containing 5% TPA, 5% TPA plus 4% Sodium bicarbonate (NaHCO3) or 1% TPA for the next 22 weeks, and then euthanized. 5% TPA treatment induced a high incidence of urinary bladder calculi and a large amount of precipitate. Though 5% TPA plus 4% Sodium bicarbonate (NaHCO3) and 1% TPA treatment did not induce urinary bladder calculi formation, they resulted in a moderate increase in urinary precipitate. Histological examination of urinary bladder revealed that MNU-5% TPA treatment resulted in a higher incidence of simple hyperplasia, papillary or nodular hyperplasia (PN hyperplasia), papilloma and cancer than MNU control. MNU-5% TPA plus 4% Sodium bicarbonate (NaHCO3) and 1% TPA treatment increased slightly the incidence of simple hyperplasia and PN hyperplasia (not statistically significant). The major elements of the precipitate are phosphorus, potassium, sulfur, chloride, calcium and TPA. The present study indicated that the calculi induced by TPA had a strong promoting activity on urinary bladder carcinogenesis and the precipitate containing calcium terephthalate (CaTPA) may also have weak promoting activity on urinary bladder carcinogenesis.

Effects of fenvalerate on progesterone production in cultured rat granulosa cells.

In this study, primary serum-free cultured rat granulosa cells (rGCs) were used as a cellular model to investigate the effects of fenvalerate on progesterone production. Various concentrations (0, 1, 5, 25, 125 and 625 microM) of fenvalerate were added to the cell cultures for 24 h. rGCs were stimulated by compounds such as follicle-stimulating hormone (FSH), 8-bromo-cAMP or 22(R)-hydroxycholesterol (22R-HC). Progesterone production and intracellular cAMP content were measured in control and treated groups. Expression of P450 side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory protein (StAR) were monitored by real-time PCR and Western blotting. Results showed that fenvalerate inhibited basal progesterone production in rGCs in the absence of stimulators. This inhibition was stronger in the presence of FSH and was not fully reversed by 8-bromo-cAMP or 22R-HC. The increase of cAMP content, stimulated by FSH, was inhibited by fenvalerate implicating that the intracellular cAMP-dependent signal pathway was involved. Fenvalerate reduced mRNA and protein expression of P450scc. These results suggested that multi-site inhibition of progesterone production by fenvalerate including a cAMP-dependent protein kinase pathway and reduction on P450scc gene expression and/or its enzymatic activity in rGCs.

Regulation of a novel cell differentiation-associated gene, JWA during oxidative damage in K562 and MCF-7 cells.

Oxidative stress, or the production of oxygen-centered free radicals, has been hypothesized as the major source of DNA damage that can lead to a variety of diseases including cancer. It is known that 8-hydroxy-deoxyguanosine (8-oxo-dG) is a useful biomarker of oxidative DNA damage. Our recent data showed that JWA, initially being cloned as a novel cell differentiation-associated gene, was also actively responsive to environmental stressors, such as heat-shock, oxidative stress and so on. In the present study, we have applied a modified comet assay and bacterial repair endonucleases system (endonuclease III and formamidopyrimidine glycosylase) to investigate if JWA is involved in hydrogen peroxide (H2O2)-induced DNA damage and repair in K562 and MCF-7 cells, and to demonstrate if the damage is associated with 8-oxo-dG. The results from the comet assay have shown that the average tail length and the percentage of the cells with DNA tails are greatly induced by H2O2 treatment and further significantly enhanced by the post-treatment of repair endonucleases. The H2O2-induced 8-oxo-dG formation in K562 and MCF-7 cells is dose-dependent. In addition, the data have clearly demonstrated that JWA gene expression is actively induced by H2O2 treatment in K562 and MCF-7 cells. The results suggest that JWA can be regulated by oxidative stress and is actively involved in the signal pathways of oxidative stress in the cells.

JWA--a novel environmental-responsive gene, involved in estrogen receptor-associated signal pathway in MCF-7 and MDA-MB-231 breast carcinoma cells.

The pyrethroid insecticide fenvalerate and the organophosphorus insecticide phoxim are now the most widely used agents for indoor pest control in China. Fenvalerate was shown to mimic estrogenic activity, whereas phoxim did not induce similar effects. Our previous studies demonstrated that JWA, a novel retinoic acid-inducible and cytoskeleton-associated gene, is also a potential environmental-responsive gene with increased expression to oxidative and heat-shock stresses. In the present study, the influence of both fenvalerate and phoxim was examined on the expression of JWA in MCF-7 (ER+) and MDA-MB-231 (ER-) human breast carcinoma cell lines. Concentrations of 0.01, 1, and 100 micromol/L of fenvalerate or phoxim were selected to treat both MCF-7 and MDA-MB-231 cells at 1, 3, and 5 d, respectively. The MTT results only showed that fenvalerate stimulated MCF-7 cell proliferation. Western blot assay was employed to detect the expressions of JWA and heat-shock proteins (hsp27 and hsp70). The results showed that after treatment with fenvalerate, both JWA and hsp70 showed similar expression patterns in the both cell lines; however, all the expression patterns of JWA, hsp27, and hsp70 were evidently reversed between ER+ and ER- cells. In addition, phoxim-treated cells showed a concentration-dependent relationship in JWA expression at all time points. These results suggest that JWA has similar functions with respect to hsp27 and hsp70, and might be a novel signal molecule in estrogen receptor-related signal transduction pathways in mammalian cells.

The roles of metallothionein on cadmium-induced testes damages in Sprague-Dawley rats.

The present study was to investigate whether metallothionein (MT) was involved in sensitivity of testis to cadmium (Cd) and protection of rats from Cd-induced testis damages. The rats were treated by intraperitoneal injection with 0.2, 0.4, and 0.8mg Cd/kg BW for 7 days. The atomic absorption spectrophotometry and cadmium hemoglobin affinity assay were applied to evaluate the contents of Cd and MT in testis and liver. The testis glutathione (GSH), malondialdehyde (MDA) and daily sperm production were measured. There were substantial increases of both Cd and MT in the liver after Cd exposure. The testis Cd and MT contents were lower than those in the corresponding liver in Cd-exposed rats. Low doses of Cd (0.2 and 0.4mg/kg BW) induced MT in testis, while a significant decline of MT was found in rats treated with 0.8mg Cd/kg BW. By a concomitant decrease of MT, there was an obvious increase of MDA and marked decreases of GSH, daily sperm production in rats treated with 0.8mg Cd/kg BW. These findings suggested MT was more difficult to be induced in the testis than in the liver by Cd, which might account for the high susceptibility of testis to Cd. MT, increased by a low dose of Cd, played an important role in protecting testis against Cd toxicity by sequestering and antioxidating.

Terephthalic acid occupational exposure and its effect on organ functions in fiber workers.

OBJECTIVES: : To investigate the exposure to terephthalic acid (TPA), and to evaluate it's effects on organ function including the potential risk factors for uroliths and bladder tumor to TPA. METHODS: : Exposure-response modeling was carried out in a cohort of 141 TPA exposure workers and three subgroups were classified according to their urine TPA concentration. The control group consisted of 77 workers with no exposure to TPA dust. The inhalatory exposure of the application workers was estimated from biological monitoring data. Urine and blood samples were collected from all workers before and after work shift to monitor variables of liver, kidney, and lung. Haematological variables and serum biochemistry were valued, pulmonary functions were tested, and ion changes in both serum and urine were measured. RESULTS: : Increased urinary excretion of TPA (0-5mmol/molCr after the shift) reflected occupational workers TPA exposure. We also observed the exposure-response relations for the intensity of TPA exposure and the urine variables. Increased serum aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) but within normal range is not increased. The slightly increased serum angiotensin-converting enzyme activity (SACE) was considered to be related to particulate of airborne TPA dust inhalation. No difference between referents and workers exposed to TPA was found for haematological variables. CONCLUSIONS: : No clinical organ dysfunctions were found in this investigation working with TPA. However, special precautions are still necessarily taken to avoid excessive or prolonged contact.

Alterations of FSH-stimulated progesterone production and calcium homeostasis in primarily cultured human luteinizing-granulosa cells induced by fenvalerate.

Fenvalerate, a synthetic pyrethroid, is widely used in agriculture and other domestic applications in China. Recently, Fenvalerate has been suspected to be one of the endocrine-disrupting chemicals (EDC). In this study, we investigated the effects of fenvalerate on follicle-stimulating hormone (FSH)-stimulated progesterone (P4) production by human ovarian luteinizing-granulosa cells (hGLCs). After 24 h incubation, fenvalerate inhibited FSH-stimulated P4 production. At the same time, FSH-stimulated cAMP also decreased. Due to calcium and Ca2+ -calmodulin (CaM) system involving gonadotropin-stimulated steroidogenesis by granulosa cells, we then evaluated the effects of fenvalerate on trifluoperazine (TFP)- and verapamil-driven FSH-stimulated P4 production. The results showed that calcium or calmodulin might play a role in fenvalerate-induced alterations in FSH-stimulated P4 biosynthesis. Then, the effects of fenvalerate on calcium homeostasis in hGLCs were studied. The result showed that 5 microM fenvalerate induced a slow increase in [Ca2+]i in hGLCs by using a fluorescent Ca2+ indicator fluo-3/AM. The changes in total concentration of CaM in hGLCs induced by fenvalerate were evaluated by a method of immunofluorescence. There is a significant increase in all treated groups. In summary, fenvalerate could inhibit FSH-stimulated P4 production. Also, fenvalerate interferes with calcium homeostasis in hGLCs. The effects of fenvalerate on FSH-stimulated ovarian steroidogenesis may be mediated partly through calcium signal.

Inactivation of thyroid peroxidase by soy isoflavones, in vitro and in vivo.

Soy-containing foods and dietary supplements are widely consumed for putative health benefits (e.g. cancer chemoprevention, beneficial effects on serum lipids associated with cardiovascular health, reduction of osteoporosis, relief of menopausal symptoms). However, studies of soy isoflavones in experimental animals suggest possible adverse effects as well (e.g. enhancement of reproductive organ cancer, modulation of endocrine function, anti-thyroid effects). This paper reviews the evidence in humans and animals for anti-thyroid effects of soy and its principal isoflavones, genistein and daidzein.

Mass spectrometric determination of p-nonylphenol metabolism and disposition following oral administration to Sprague-Dawley rats.

Isomers of 4-nonylphenol (NP), which are important industrial compounds and environmental breakdown products from widely used surfactants, have estrogenic activity in vitro and in vivo that has prompted interest in its potential for modulation of endocrine function in humans and wildlife. Mass spectrometry was used to quantify NP and metabolites in serum and endocrine-responsive tissues from dietary exposure in Sprague-Dawley rats. Tissue accumulation of NP aglycone was observed despite the predominance of glucuronidation in blood. Serum toxicokinetics of total NP, measured following gavage administration, showed rapid absorption and elimination (average half-times 0.8 and 3.5 h, respectively). NP was similarly administered by gavage to pregnant dams and total and aglycone NP were measured in dam serum and fetuses to show placental transfer into serum and brain. These data provide a basis for future correlations of biologic effects observed following dietary exposure in rats with those predicted from environmental exposures to humans.