Clofibrate, a Peroxisome Proliferator-Activated Receptor-Alpha (PPARα) Agonist, and Its Molecular Mechanisms of Action against Sodium Fluoride-Induced Toxicity.

Sodium fluoride (NaF) is one of the neglected environmental pollutants. It is ubiquitously found in the soil, water, and environment. Interestingly, fluoride has been extensively utilized for prevention of dental caries and tartar formation, and may be added to mouthwash, mouth rinse, and toothpastes. This study is aimed at mitigating fluoride-induced hypertension and nephrotoxicity with clofibrate, a peroxisome proliferator-activated receptor-alpha (PPARα) agonist. For this study, forty male Wistar rats were used and randomly grouped into ten rats per group, control, sodium fluoride (NaF; 300 ppm) only, NaF plus clofibrate (250 mg/kg) and NaF plus lisinopril (10 mg/kg), respectively, for 7 days. The administration of NaF was by drinking water ad libitum, while clofibrate and lisinopril were administered by oral gavage. Administration of NaF induced hypertension, and was accompanied with exaggerated oxidative stress; depletion of antioxidant defence system; reduced nitric oxide production; increased systolic, diastolic and mean arterial pressure; activation of angiotensin-converting enzyme activity and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB); and testicular apoptosis. Treatment of rats with clofibrate reduced oxidative stress, improved antioxidant status, lowered high blood pressure through the inhibition of angiotensin-converting enzyme activity, mineralocorticoid receptor over-activation, and abrogated testicular apoptosis. Taken together, clofibrate could offer exceptional therapeutic benefit in mitigating toxicity associated with sodium fluoride.

Reduced hepatic global hydroxymethylation in mice treated with non-genotoxic carcinogens is transiently reversible with a methyl supplemented diet.

Non-genotoxic carcinogens (NGCs) are known to cause perturbations in DNA methylation, which can be an early event leading to changes in gene expression and the onset of carcinogenicity. Phenobarbital (PB) has been shown to alter liver DNA methylation and hydroxymethylation patterns in mice in a time dependent manner. The goals of this study were to assess if clofibrate (CFB), a well-studied rodent NGC, would produce epigenetic changes in mice similar to PB, and if a methyl donor supplementation (MDS) would modulate epigenetic and gene expression changes induced by phenobarbital. CByB6F1 mice were treated with 0.5% clofibrate or 0.14% phenobarbital for 7 and 28 days. A subgroup of PB treated and control mice were also fed MDS diet. Liquid Chromatography-Ionization Mass Spectrometry (LC-MS) was used to quantify global liver 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) levels. Gene expression analysis was conducted using Affymetrix microarrays. A decrease in liver 5hmC but not 5mC levels was observed upon treatment with both CFB and PB with varying time of onset. We observed moderate increases in 5hmC levels in PB-treated mice when exposed to MDS diet and lower expression levels of several phenobarbital induced genes involved in cell proliferation, growth, and invasion, suggesting an early modulating effect of methyl donor supplementation. Overall, epigenetic profiling can aid in identifying early mechanism-based biomarkers of non-genotoxic carcinogenicity and increases the quality of cancer risk assessment for candidate drugs. Global DNA methylation assessment by LC-MS is an informative first step toward understanding the risk of carcinogenicity.

Pilot studies evaluating the nongenotoxic rodent carcinogens phenobarbital and clofibrate in the rat Pig-a assay.

The Pig-a assay is an emerging and promising in vivo method to determine mutagenic potential of chemicals. Since its development in 2008, remarkable progress has been made in harmonizing and characterizing the test procedures, primarily using known mutagenic chemicals. The purpose of the present study was to evaluate specificity of the Pig-a assay using two nongenotoxic and well-characterized rodent liver carcinogens, phenobarbital and clofibrate, in male F344/DuCrl rats. Daily oral administration of phenobarbital or clofibrate at established hepatotoxic doses for 28 days resulted in substantial hepatic alterations, however, did not increase the frequency of Pig-a mutation markers (RETCD59- and RBCCD59- ) compared to vehicle control or pre-exposure (Day -5) mutant frequencies. These results are consistent with the existing literature on the nonmutagenic mode of action (MoA) of phenobarbital and clofibrate liver tumors. The present study contributes to the limited, but expanding evidence on the specificity of the Pig-a assay and further for the investigations of carcinogenic MoAs, i.e., mutagenic or nonmutagenic potential of chemicals. Environ. Mol. Mutagen. 60:42-46, 2019. © 2018 Wiley Periodicals, Inc.

Applying the erythrocyte Pig-a assay concept to rat epididymal sperm for germ cell mutagenicity evaluation.

The Pig-a assay, a recently developed in vivo somatic gene mutation assay, is based on the identification of mutant erythrocytes that have an altered repertoire of glycosylphosphatidylinositol (GPI)-anchored cell surface markers. We hypothesized that the erythrocyte Pig-a assay concept could be applied to rat cauda epididymal spermatozoa (sperm) for germ cell mutagenicity evaluation. We used GPI-anchored CD59 as the Pig-a mutation marker and examined the frequency of CD59-negative sperm using flow cytometry. A reconstruction experiment that spiked un-labeled sperm (mutant-mimic) into labeled sperm at specific ratios yielded good agreement between the detected and expected frequencies of mutant-mimic sperm, demonstrating the analytical ability for CD59-negative sperm detection. Furthermore, this methodology was assessed in F344/DuCrl rats administered N-ethyl-N-nitrosourea (ENU), a prototypical mutagen, or clofibrate, a lipid-lowering drug. Rats treated with 1, 10, or 20 mg/kg body weight/day (mkd) ENU via daily oral garage for five consecutive days showed a dose-dependent increase in the frequency of CD59-negative sperm on study day 63 (i.e., 58 days after the last ENU dose). This ENU dosing regimen also increased the frequency of CD59-negative erythrocytes. In rats treated with 300 mkd clofibrate via daily oral garage for consecutive 28 days, no treatment-related changes were detected in the frequency of CD59-negative sperm on study day 85 (i.e., 57 days after the last dose) or in the frequency of CD59-negative erythrocytes on study day 29. In conclusion, these data suggest that the epidiymal sperm Pig-a assay in rats is a promising method for evaluating germ cell mutagenicity. Environ. Mol. Mutagen. 58:485-493, 2017. © 2017 Wiley Periodicals, Inc.

Dose-response analysis of epigenetic, metabolic, and apical endpoints after short-term exposure to experimental hepatotoxicants.

Identification of sensitive and novel biomarkers or endpoints associated with toxicity and carcinogenesis is of a high priority. There is increasing interest in the incorporation of epigenetic and metabolic biomarkers to complement apical data; however, a number of questions, including the tissue specificity, dose-response patterns, early detection of those endpoints, and the added value need to be addressed. In this study, we investigated the dose-response relationship between apical, epigenetic, and metabolomics endpoints following short-term exposure to experimental hepatotoxicants, clofibrate (CF) and phenobarbital (PB). Male F344 rats were exposed to PB (0, 5, 25, and 100 mg/kg/day) or CF (0, 10, 50, and 250 mg/kg/day) for seven days. Exposure to PB or CF resulted in dose-dependent increases in relative liver weights, hepatocellular hypertrophy and proliferation, and increases in Cyp2b1 and Cyp4a1 transcripts. These changes were associated with altered histone modifications within the regulatory units of cytochrome genes, LINE-1 DNA hypomethylation, and altered microRNA profiles. Metabolomics data indicated alterations in the metabolism of bile acids. This study provides the first comprehensive analysis of the apical, epigenetic and metabolic alterations, and suggests that the latter two occur within or near the dose response curve of apical endpoint alterations following exposure to experimental hepatotoxicants.

Quantitative weight of evidence to assess confidence in potential modes of action.

The evolved World Health Organization/International Programme on Chemical Safety mode of action (MOA) framework provides a structure for evaluating evidence in pathways of causally linked key events (KE) leading to adverse health effects. Although employed globally, variability in use of the MOA framework has led to different interpretations of the sufficiency of evidence in support of hypothesized MOAs. A proof of concept extension of the MOA framework is proposed for scoring confidence in the supporting data to improve scientific justification for MOA use in characterizing hazards and selecting dose-response extrapolation methods for specific chemicals. This involves selecting hypothesized MOAs, and then, for each MOA, scoring the weight of evidence (WOE) in support of causality for each KE using evolved Bradford Hill causal considerations (biological plausibility, essentiality, dose-response concordance, consistency, and analogy). This early proof of concept method is demonstrated by comparing two potential MOAs (mutagenicity and peroxisome proliferator activated receptor-alpha) for clofibrate, a rodent liver carcinogen. Quantitative confidence scoring of hypothesized MOAs is shown to be useful in characterizing the likely operative MOA. To guide method refinement and future confidence scoring for a spectrum of MOAs, areas warranting further focus and lessons learned, including the need to incorporate a narrative discussion of the weights used in the evaluation and an overall evaluation of the plausibility of the outcome, are presented.

Biomarker evaluation of skeletal muscle toxicity following clofibrate administration in rats.

The use of sensitive biomarkers to monitor skeletal muscle toxicity in preclinical toxicity studies is important for the risk assessment in humans during the development of a novel compound. Skeletal muscle toxicity in Sprague Dawley Rats was induced with clofibrate at different dose levels for 7 days to compare standard clinical pathology assays with novel skeletal muscle and cardiac muscle biomarkers, gene expression and histopathological changes. The standard clinical pathology assays aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatine kinase (CK) enzyme activity were compared to novel biomarkers fatty acid binding protein 3 (Fabp3), myosin light chain 3 (Myl3), muscular isoform of CK immunoreactivity (three isoforms CKBB, CKMM, CKMB), parvalbumin (Prv), skeletal troponin I (sTnI), cardiac troponin T (cTnT), cardiac troponin I (cTnI), CKMM, and myoglobin (Myo). The biomarker elevations were correlated to histopathological findings detected in several muscles and gene expression changes. Clofibrate predominantly induced skeletal muscle toxicity of type I fibers of low magnitude. Useful biomarkers for skeletal muscle toxicity were AST, Fabp3, Myl3, (CKMB) and sTnI. Measurements of CK enzyme activity by a standard clinical assay were not useful for monitoring clofibrate-induced skeletal muscle toxicity in the rat at the doses used in this study.

Estrogen and androgen receptor status in hepatocellular hypertrophy induced by phenobarbital, clofibrate, and piperonyl butoxide in F344 rats.

The present study examined hepatic estrogen receptor (ER) and androgen receptor (AR) levels as well as estrogen-signaling status in a model of rat hepatic hypertrophy induced by phenobarbital (PB), chlofibrate (CF), or piperonyl butoxide (PBO). Male F344 rats were fed with PB at 2,500 ppm, CF at 2,500 ppm, and PBO at 20,000 ppm for 3 days, 4 weeks, and 13 weeks. CF and PBO induced diffuse hypertrophy, while centrilobular hypertrophy was observed with PB administration. The levels of mRNA for ERα, AR and leukemia inhibitory factor receptor (LIFR) which was found to be estrogen responsive in the present study, were determined by quantitative RT-PCR. In the CF and PBO groups, ERα mRNA expression was reduced, and consequently, the expression of a responsive gene, LIFR, was also decreased, while PB had no effect on ER mRNA levels. AR mRNA expression decreased in all the treated groups, but reduction was persistent only in PB group. Recently, LIFR was identified as a tumor suppressor gene in human HCC. Thus, LIFR may be one of the key mediators of hepatic carcinogenesis induced by CF and PBO, but PB appears to act via different mechanisms.

Super-induced gene expression of the N-methyl-D-aspartate receptor 2C subunit in chemical-induced hypertrophic liver in rats.

To identify gene expression that can be closely involved in chemical-induced hepatocellular hypertrophy, the hepatic gene expression profile was assessed by cDNA microarray analysis in male F344 rats fed for 3 days, 4 weeks, and 13 weeks a diet containing a hepatocellular hypertrophy inducer, either phenobarbital (500 ppm), clofibrate (2,500 ppm), or piperonyl butoxide (20,000 ppm). The results showed that, in all treatment groups, the increased expressional rate of the Grin2c gene, which encodes the N-methyl-D-aspartate receptor 2C subunit (NR2C), was the highest among those of all the genes tested, as compared with the corresponding gene expression in rats fed a normal diet. Moreover, real-time RT-PCR analysis showed that the expression levels of the Grin2c gene in rats fed with each chemical clearly increased in a chemical treatment period-dependent fashion, and that the increased rate was closely correlated with the grade of hypertrophy of hepatocytes rather than with the increased rate in liver weight. These results suggest the possibility that chemical-induced NR2C expression relates to the development of hepatocellular hypertrophy.

Altered expression of GADD45 genes during the development of chemical-mediated liver hypertrophy and liver tumor promotion in rats.

The purpose of our study was to examine the altered gene expression associated with nongenotoxic chemical-mediated liver hypertrophy and successive liver tumor promotion. Five-week-old male rats were fed a basal diet or a diet containing phenobarbital (PB) or clofibrate (CF) for 3 days, 4 weeks, and 13 weeks. Hepatic expression profiling of cell growth- and stress-related genes, as well as those involved in xenobiotic metabolism, was performed by DNA microarray and/or real time quantitative reverse transcription-polymerase chain reaction. The induction of liver hypertrophy and hepatic cytochrome P450 (CYP) isoforms (CYP2B1/2B2 for PB and CYP4A1 for CF) by PB and CF were clearly observed at all the treatment periods examined. Genes encoding DNA damage-inducible 45 (GADD45) family proteins, in particular GADD45g (GADD45 gamma) were down-regulated by treatment with either PB or CF for 4 and 13 weeks. The chemical-mediated development of liver hypertrophy, induction of hepatic CYPs, and suppression of hepatic GADD45g gene at week 13 disappeared at 4 weeks following cessation of the chemical treatment. Additionally, DNA microarray data indicated that cell cycle-related genes such as cyclins CCNB1 and CCNA2 and cyclin-dependent kinase inhibitor CDKN3 were also down-regulated by treatment with either PB or CF at 13 weeks. Since GADD45 functions as a chemical and radiation stress sensor by interacting with cyclins and cyclin-dependent kinase inhibitors, the decrease in the gene expression of GADD45g mRNA observed in this study, may be associated with nongenotoxic chemical-induced tumor promotion of hepatocarcinogenesis rather than liver hypertrophy.

Can pharmaceuticals interfere with the synthesis of active androgens in male fish? An in vitro study.

The in vitro interference of fibrate (gemfibrozil, clofibrate, clofibric acid), anti-inflammatory (ibuprofen, diclofenac), and anti-depressive (fluoxetine, fluvoxamine) drugs with key enzymatic activities-C17,20-lyase and CYP11ß-involved in the synthesis of active androgens in gonads of male carp have been investigated. Among the tested compounds, fluvoxamine and fluoxetine were the strongest inhibitors of C17,20-lyase and CYP11ß enzymes, with IC50s in the range of 321-335 µM and 244-550 µM, respectively. To our knowledge this is the first report on the interaction of pharmaceutical compounds with enzymatic systems involved in the synthesis of oxy-androgens. As oxy-androgens are known to influence spermatogenesis and stimulate reproductive behavior and secondary sexual characteristics in male fish, this work highlights the need for further investigating these endpoints when designing specific in vivo studies to assess the endocrine disruptive effect of pharmaceuticals in fish.

Decrease of hepatic stellate cells in rats with enhanced sensitivity to clofibrate-induced hepatocarcinogenesis.

To examine the possible involvement of nonparenchymal cells in the development of preneoplastic hepatic lesions induced by clofibrate (CF), alterations of these cells were investigated immunohistochemically in glutathione S-transferase M1 gene polymorphic rats (KS and NC types) with different cancer susceptibilities. After CF administration for 8 weeks, α-smooth muscle actin (α-SMA)-positive hepatic stellate cells (HSC) were markedly decreased in sensitive KS-type rats, but not in the NC-type rats. Kupffer cells were decreased with similar extents between them. The sinusoidal endothelial cells were not changed in either type. The other markers for HSC, vimentin and CRBP1, also confirmed the decrease of HSC in the KS type. The decrease of HSC was not observed at 4 weeks of CF administration. Preneoplastic peroxisomal bifunctional enzyme-negative foci were detected in the KS-type rats at 8 weeks of CF administration, but not at 4 weeks. Human HSC were cultured in the presence of clofibric acid and expression of most HSC marker genes, such as vimentin and α-SMA (ACTA2), evaluated by a microarray, was not altered by the treatment, suggesting that HSC loss in the KS-type rats was not due to the direct toxic effect of CF. The expression levels of most HSC marker genes were low in both control and CF-treated rat livers. A possible link between HSC loss and the development of preneoplastic hepatic foci is discussed.

26-Week carcinogenicity study of di-isodecyl phthalate by dietary administration to CB6F1-rasH2 transgenic mice.

This study examined the carcinogenic potential of di-isodecyl phthalate (DIDP) in rasH2 mice. DIDP was administered to 15 rasH2 mice/gender/group at dietary levels of 0, 0.1, 0.33, or 1% and 15 wild-type mice/gender/group at dietary levels of 0 and 1% for 26 weeks. Non-neoplastic changes were observed in the liver (parenchymal inflammation, fatty changes, diffuse hepatocyte hypertrophy with eosinophilic granules and focal necrosis) and kidneys (tubular basophilia and tubular hyperplasia) after administration of DIDP in the rasH2 and wild-type mice. In the neoplastic lesions, there were a higher number of hepatocellular adenomas in the male rasH2 mice receiving 1% DIDP, compared with the findings in the liver of control rasH2 mice or wild-type mice. The incidence of hepatocellular adenomas in the 0.1, 0.33, and 1% DIDP exposed rasH2 mice was 7% (1/15), 7% (1/15), and 33% (5/15), respectively. This study adds a set of results for an additional test chemical for the performance of the rasH2 short-term transgenic model to the existing database of 3 compounds (WY-14643, DEHP, and clofibrate) tested in the ILSI/HESI ACT project.

Expression of two cytochrome P450 aromatase genes is regulated by endocrine disrupting chemicals in rare minnow Gobiocypris rarus juveniles.

To elucidate the effects of endocrine disrupting chemicals (EDCs) on aromatase, the rare minnow ovarian and brain P450 aromatase (cyp19a1a and cyp19a1b) cDNA and their 5'-flanking regions were isolated and characterized. RT-PCR analysis revealed that the rare minnow cyp19a1a mRNA was predominantly expressed in ovary while cyp19a1b was predominantly expressed in brain. Sequences for binding sites of steroidogenic factor-1, peroxisome proliferators-activated receptor, aryl hydrocarbon receptor, CCAAT/enhancer binding protein, estrogen responsive element, glucocorticoid responsive element, and retinoic acid receptor were identified on promoter regions of cyp19a1 genes. The influence of several EDCs on the transcript abundance of cyp19a1a and cyp19a1b was investigated in rare minnow juveniles. Clofibrate did not influence the expression of either cyp19a1 genes. Exposure to 1nM ethinylestradiol (EE2) for 3days significantly downregulated the expression of cyp19a1a gene, however 0.1 and 1 nM EE2 significantly increased the gene expression of cyp19a1b. Exposure to 100 and 1000 nM 4-nonylphenol (NP) significantly suppressed the cyp19a1a expression, but it had no effect on the expression of cyp19a1b gene. Bisphenol A (BPA) strongly suppressed the cyp19a1b gene expression from 0.1 to 10 nM and significantly suppressed the gene expression of cyp19a1a only at 10 nM. These results indicate that EDCs may influence the expression of cyp19a1 genes through differential transcriptional modulation in rare minnow juveniles.

Proteomics analysis of cardiac muscle from rats with peroxisomal proliferator-activated receptor alpha (PPARalpha) stimulation.

To investigate peroxisomal proliferator-activated receptor alpha (PPARalpha) signal responses in heart muscle, we performed LC-MS/MS-based proteomics analysis of heart muscle from rats given fenofibrate or clofibrate. Fenofibrate increased the expression of ACAA2, DECR1, and ECH1 consistent with activation of PPARalpha. Fenofibrate and clofibrate reduced the expression of 10 and 12 proteins, respectively with the expression of ACSL1, SLC25A4, A1BG, HADHA, ATP2A2, BDH1, ETFDH, HADHB, and CPT2 being reduced in common with both of fibrate-treated groups. The approach adopted in this study provides an efficient method for monitoring global changes in protein expression.

Glutathione S-transferase A4 is a positive marker for rat hepatic foci induced by clofibrate and genotoxic carcinogens.

Peroxisome proliferators (PP), including clofibrate (CF), are non-genotoxic rodent carcinogens, and oxidative DNA damages are suggested as a causative event for carcinogenesis. Gene expression profiles differ between hepatic lesions induced by PP and genotoxic carcinogens. Our previous study revealed that expression of L-bifunctional enzyme (enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase, BE) was repressed in preneoplastic lesions induced by PP, whereas it was enhanced in the surrounding tissues. In the present study, we immunohistochemically examined expression of the specific glutathione S-transferase (GST) form, GST-A4, which detoxifies 4-hydroxy-alkenal, the end-product of lipid peroxides, and nuclear factor-erythroid 2-related factor 2 (Nrf2), a transcription factor for many genes encoding drug-metabolizing enzymes and defending enzymes against oxidative stress, during rat hepatocarcinogenesis induced by CF and genotoxic carcinogens. GST-A4 and Nrf2 were not expressed in BE-negative foci at 8 weeks of CF administration, but were expressed in the foci at 60 weeks. GST-A4-positive foci appeared at later stages than BE-negative foci, but its localization was coincidental with that of the latter foci. The areas of GST-A4-positive foci were larger than those of BE-negative foci without GST-A4 expression. Most GST-A4-positive foci were also positive for Nrf2. In rat livers induced by genotoxic carcinogens, GST-P-negative foci as well as GST-P-positive foci were demonstrated. GST-A4 and Nrf2 were expressed in GST-P-negative foci, whereas they were not expressed in most GST-P-positive foci. Thus, GST-A4-positive foci developed in rat livers by CF and genotoxic carcinogen administration, indicating that the enzyme is a positive marker for hepatic foci induced by these different carcinogens.

Characterization of peroxisome proliferator-activated receptor alpha--independent effects of PPARalpha activators in the rodent liver: di-(2-ethylhexyl) phthalate also activates the constitutive-activated receptor.

Peroxisome proliferator chemicals (PPC) are thought to mediate their effects in rodents on hepatocyte growth and liver cancer through the nuclear receptor peroxisome proliferator-activated receptor (PPAR) alpha. Recent studies indicate that the plasticizer di-(2-ethylhexyl) phthalate (DEHP) increased the incidence of liver tumors in PPARalpha-null mice. We hypothesized that some PPC, including DEHP, induce transcriptional changes independent of PPARalpha but dependent on other nuclear receptors, including the constitutive-activated receptor (CAR) that mediates phenobarbital (PB) effects on hepatocyte growth and liver tumor induction. To determine the potential role of CAR in mediating effects of PPC, a meta-analysis was performed on transcript profiles from published studies in which rats and mice were exposed to PPC and compared the profiles to those produced by exposure to PB. Valproic acid, clofibrate, and DEHP in rat liver and DEHP in mouse liver induced genes, including Cyp2b family members that are known to be regulated by CAR. Examination of transcript changes by Affymetrix ST 1.0 arrays and reverse transcription-PCR in the livers of DEHP-treated wild-type, PPARalpha-null, and CAR-null mice demonstrated that (1) most (approximately 94%) of the transcriptional changes induced by DEHP were PPARalpha-dependent, (2) many PPARalpha-independent genes overlapped with those regulated by PB, (3) induction of genes Cyp2b10, Cyp3a11, and metallothionine-1 by DEHP was CAR dependent but PPARalpha-independent, and (4) induction of a number of genes (Cyp8b1, Gstm4, and Gstm7) was independent of both CAR and PPARalpha. Our results indicate that exposure to PPARalpha activators including DEHP leads to activation of multiple nuclear receptors in the rodent liver.

Scoring multiple toxicological endpoints using a toxicogenomic database.

As information regarding microarray data sets and toxicogenomic biomarkers grows rapidly, the process of analyzing data and interpreting the results is increasingly complicated. To facilitate data analysis, a simple expression ratio-based scoring method called the TGP1 score was previously proposed [Kiyosawa, N., Shiwaku, K., Hirode, M., Omura, K., Uehara, T., Shimizu, T., Mizukawa, Y., Miyagishima, T., Ono, A., Nagao, T., Urushidani, T., 2006. Utilization of a one-dimensional score for surveying chemical-induced changes in expression levels of multiple biomarker gene sets using a large-scale toxicogenomics database. J. Toxicol. Sci. 31, 433-448]. Although the TGP1 score has demonstrated its efficacy for rapid comprehension of large-scale toxicogenomic data sets, inclusion of low quality gene expression data in the biomarker gene set produced flaws in the calculated score. To overcome this shortcoming, we tested a new scoring method called the differentially expressed gene score (D-score), where Detection Call as well as signal log ratios generated by MAS5 algorithm on Affymetrix GeneChip data were considered for the calculation. Four prototypical toxicants, namely acetaminophen, phenobarbital, clofibrate and acetamidofluorene, were used for detailed analysis. A toxicogenomics database (TG-GATEs) was utilized as a reference data set. The D-score successfully alleviated the effects of low quality data on the score calculation, and captured the overall direction of expression changes as well as the magnitude of expression change level of a set of genes, highlighting the affected toxicological endpoints elicited by chemical treatment. The D-score will be useful for high-throughput toxicity screening using a toxicogenomic database and biomarkers.

Genomic profiling reveals an alternate mechanism for hepatic tumor promotion by perfluorooctanoic acid in rainbow trout.

BACKGROUND: Perfluorooctanoic acid (PFOA) is a potent hepatocarcinogen and peroxisome proliferator (PP) in rodents. Humans are not susceptible to peroxisome proliferation and are considered refractory to carcinogenesis by PPs. Previous studies with rainbow trout indicate they are also insensitive to peroxisome proliferation by the PP dehydroepiandrosterone (DHEA), but are still susceptible to enhanced hepatocarcinogenesis after chronic exposure. OBJECTIVES: In this study, we used trout as a unique in vivo tumor model to study the potential for PFOA carcinogenesis in the absence of peroxisome proliferation compared with the structurally diverse PPs clofibrate (CLOF) and DHEA. Mechanisms of carcinogenesis were identified from hepatic gene expression profiles phenotypically anchored to tumor outcome. METHODS: We fed aflatoxin B(1) or sham-initiated animals 200-1,800 ppm PFOA in the diet for 30 weeks for tumor analysis. We subsequently examined gene expression by cDNA array in animals fed PFOA, DHEA, CLOF, or 5 ppm 17beta-estradiol (E(2), a known tumor promoter) in the diet for 14 days. RESULTS: PFOA (1,800 ppm or 50 mg/kg/day) and DHEA treatments resulted in enhanced liver tumor incidence and multiplicity (p < 0.0001), whereas CLOF showed no effect. Carcinogenesis was independent of peroxisome proliferation, measured by lack of peroxisomal beta-oxidation and catalase activity. Alternately, both tumor promoters, PFOA and DHEA, resulted in estrogenic gene signatures with strong correlation to E(2) by Pearson correlation (R = 0.81 and 0.78, respectively), whereas CLOF regulated no genes in common with E(2). CONCLUSIONS: These data suggest that the tumor-promoting activities of PFOA in trout are due to novel mechanisms involving estrogenic signaling and are independent of peroxisome proliferation.

Toxicokintic and toxicodynamic analysis of clofibrate based on free drug concentrations in nagase analbuminemia rats (NAR).

Toxicokinetics (TK) is usually performed by measurement of the total drug concentrations in plasma. However, free drug concentrations in plasma are considered to correlate directly with toxicodynamics (TD). In the present study, to evaluate the applicability of TK/TD analysis based on free drug concentrations, we investigated the TK/TD of clofibrate, which binds to albumin with a higher ratio, using an albumin-deficient mutant strain, Nagase analbuminemia rats (NAR). TK, blood chemistry, histopathology, drug and fatty acid metabolizing enzymes and microarray analysis in the liver were examined after a 4-day oral administration of clofibrate. Compared to Sprague-Dawley (SD) rats, the parent strain of NAR, 4.1-fold higher AUC(0-24hr) based on free drug concentrations (3445 versus 844 microg.hr/ml) was observed in NAR when both rats showed the same level of AUC(0-24hr) based on the total drug concentrations (4436 versus 4237microg.hr/ml). Additionally, more severe hepatocellular hypertrophy, increase in aspartate transaminase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH), decrease in total cholesterol (T.CHO), phospholipid (PL), triglyceride (TG), and non-esterified fatty acid (NEFA), and increase in the mRNA levels of fatty acid metabolizing enzymes (FAOS, CAT, and CPT) were observed in NAR at the same dose. These results demonstrated that NAR developed more severe toxicities and pharmacological effects than SD rats correlating with the higher AUC of the free drug concentrations. The results also suggested that TK/TD analysis based on the free drug concentration is appropriate to interpret the relationship between exposure and toxicity in cases of protein binding saturation including protein decrease or species differences on protein binding, especially when drugs showing a higher protein binding ratio are dosed.