Evaluating H295R steroidogenesis assay data for robust interpretation.

The in vitro H295R steroidogenesis assay (OECD TG 456) is used to determine a chemical's potential to interfere with steroid hormone synthesis/metabolism. As positive outcomes in this assay can trigger significant higher tiered testing, we compiled a stakeholder database of reference and test item H295R data to characterize assay outcomes. Information concerning whether a Level 5 reproductive toxicity study was triggered due to a positive outcome in the H295R assay was also included. Quality control acceptance criteria were not always achieved, suggesting this assay is challenging to conduct within the guideline specifications. Analysis of test item data demonstrated that pairwise significance testing to controls allowed for overly sensitive statistically significant positive outcomes, which likely contribute to the assay's high positive hit rate. Complementary interpretation criteria (e.g., 1.5-fold change threshold) markedly reduced the rate of equivocal and positive outcomes thus improving identification of robust positive effects in the assay. Finally, a case study (positive H295R outcome and no endocrine adversity in vivo) is presented, which suggests that stricter data interpretation criteria could refine necessary in vivo follow-up testing. Overall, the described additional criteria could improve H295R data interpretation and help inform on how to best leverage this assay for regulatory purposes.

Effects of turmeric (Curcuma longa) on the expression of hepatic genes associated with biotransformation, antioxidant, and immune systems in broiler chicks fed aflatoxin.

The objective of the present study was to evaluate the efficacy of curcumin, an antioxidant found in turmeric (Curcuma longa) powder (TMP), to ameliorate changes in gene expression in the livers of broiler chicks fed aflatoxin B(1) (AFB(1)). Four pen replicates of 5 chicks each were assigned to each of 4 dietary treatments, which included the following: A) basal diet containing no AFB(1) or TMP (control), B) basal diet supplemented with TMP (0.5%) that supplied 74 mg/kg of curcumin, C) basal diet supplemented with 1.0 mg of AFB(1)/kg of diet, and D) basal diet supplemented with TMP that supplied 74 mg/kg of curcumin and 1.0 mg of AFB(1)/kg of diet. Aflatoxin reduced (P < 0.05) feed intake and BW gain and increased (P < 0.05) relative liver weight. Addition of TMP to the AFB(1) diet ameliorated (P < 0.05) the negative effects of AFB(1) on growth performance and liver weight. At the end of the 3-wk treatment period, livers were collected (6 per treatment) to evaluate changes in the expression of genes involved in antioxidant function [catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione S-transferase (GST)], biotransformation [epoxide hydrolase (EH), cytochrome P450 1A1 and 2H1 (CYP1A1 and CYP2H1)], and the immune system [interleukins 6 and 2 (IL-6 and IL-2)]. Changes in gene expression were determined using the quantitative real-time PCR technique. There was no statistical difference in gene expression among the 4 treatment groups for CAT and IL-2 genes. Decreased expression of SOD, GST, and EH genes due to AFB(1) was alleviated by inclusion of TMP in the diet. Increased expression of IL-6, CYP1A1 and CYP2H1 genes due to AFB(1) was also alleviated by TMP. The current study demonstrates partial protective effects of TMP on changes in expression of antioxidant, biotransformation, and immune system genes in livers of chicks fed AFB(1). Practical application of the research is supplementation of TMP in diets to prevent or reduce the effects of aflatoxin in chicks fed aflatoxin-contaminated diets.

Effects of short-term heat stress on endophytic ergot alkaloid-induced alterations in rat hepatic gene expression.

Exposure to ergot alkaloids in endophyte-infected fescue (E+) is associated with impaired animal productivity, especially during heat stress, which is commonly referred to as fescue toxicosis. To elucidate the pathogenesis of this condition, the effects of short-term heat stress (HS) on hepatic gene expression in rats exposed to endophytic ergot alkaloids were evaluated. Rats implanted with telemetric transmitters to continuously measure core temperature were fed an E+ diet and maintained under thermoneutral (TN) conditions (21 degrees C) for 5 d, followed by TN or 31 degrees C (HS) conditions for 3 d. Feed intake (FI) and BW were monitored daily. The E+ and HS-induced alterations in hepatic genes were evaluated using DNA microarrays and PCR analyses. Hepatic antioxidant enzyme activities, as well as the incidence of apoptosis, were determined. As expected, intake of E+ reduced FI and BW from pretreatment levels under TN conditions, with greater reductions during short-term HS. Genes involved in gluconeogenesis and apoptosis were upregulated, whereas genes associated with oxidative phosphorylation, xenobiotic metabolism, antioxidative mechanisms, immune function, cellular proliferation, and chaperone activity were all downregulated with short-term HS. Hepatocytic apoptosis was increased and antioxidant enzyme activity decreased in the livers of rats exposed to HS. The hypothesized, exacerbating effects of HS on the direct, endophytic toxin-related and indirect, reduced caloric intake-associated alterations in hepatic gene expression were clearly demonstrated in rats and may help to elucidate the pathogenesis of fescue toxicosis in various animal species.

Toxicological and gene expression analysis of the impact of aflatoxin B1 on hepatic function of male broiler chicks.

The objective of this study was to determine the effects of dietary aflatoxin B1 (AFB1) on hepatic gene expression in male broiler chicks. Seventy-five 1-d-old male broiler chicks were assigned to 3 dietary treatments (5 replicates of 5 chicks each) from hatch to d 21. The diets contained 0, 1 and 2 mg of AFB1/kg of feed. Aflatoxin B1 reduced (P<0.05) feed intake, BW gain, serum total proteins, and serum Ca and P, but increased (P<0.01) liver weights in a dose-dependent manner. Microarray analysis was used to identify shifts in genetic expression associated with the affected physiological processes in chicks fed 0 and 2 mg of AFB1/kg of feed to identify potential targets for pharmacological/toxicological intervention. A loop design was used for microarray experiments with 3 technical and 4 biological replicates per treatment group. Ribonucleic acid was extracted from liver tissue, and its quality was determined using gel electrophoresis and spectrophotometry. High-quality RNA was purified from DNA contamination, reverse transcribed, and hybridized to an oligonucleotide microarray chip. Microarray data were analyzed using a 2-step ANOVA model and validated by quantitative real-time PCR of selected genes. Genes with false discovery rates less than 13% and fold change greater than 1.4 were considered differentially expressed. Compared with controls (0 mg of AFB1/kg), various genes associated with energy production and fatty acid metabolism (carnitine palmitoyl transferase), growth and development (insulin-like growth factor 1), antioxidant protection (glutathione S transferase), detoxification (epoxide hydrolase), coagulation (coagulation factors IX and X), and immune protection (interleukins) were downregulated, whereas genes associated with cell proliferation (ornithine decarboxylase) were upregulated in birds fed 2 mg of AFB1/kg. This study demonstrates that AFB1 exposure at a concentration of 2 mg/kg results in physiological responses associated with altered gene expression in chick livers.

Relationship of thermal status to productivity in heat-stressed dairy cows given recombinant bovine somatotropin.

The responses of lactating Holstein cows to daily administration of bovine somatotropin (bST) were measured at thermoneutrality (Tn) and under both constant and cycled heat-stress conditions to determine the relationship between thermal status and bST-induced shifts in milk production. All tests included a 5-d acclimation period at Tn (18 degrees C), followed by a 2-d increase in ambient temperature to 28.5 degrees C. After d 3, ambient temperature was cycled between 28.5 (day) and 25.5 degrees C (night) for 4 d. Daily injections with either 31 mg of bST or saline began on d 1 of the experiment. Milk production, feed intake, and respiratory rate (RR) were measured daily. Intraperitoneal, telemetric temperature transmitters were used for a continuous measure of core body temperature (T(core)). Blood samples were collected during each phase to evaluate the changes in serum chemistry in response to bST and heat stress. Following a 15-d recovery, cows were switched across injection treatments and the study was repeated. Milk production decreased by approximately 18.4% below the initial yield at Tn by the end of 7 d of heat challenge. Although a reduction in milk production occurred during heat stress in both groups, milk production was higher in bST-treated cows compared with control cows during periods of constant and cyclic heat. Likewise, bST treatment during the entire period increased the milk-to-feed ratio over the control level by approximately 11.3%. Plasma insulin-like growth factor 1 and serum nonesterified fatty acids accompanied the increased growth hormone level with bST treatment (approximately 122.0 and 88.8%, respectively), whereas plasma urea nitrogen was reduced by approximately 13.3% to reflect the shift to lipid metabolism. There was no difference in T(core) of the treatment and control groups at Tn. Both bST and control cows increased RR and T(core) above the Tn level by approximately 94.8 and 2.9%, respectively, during constant heat, with a greater increase in T(core) of bST-treated compared with control cows (approximately 0.6%). The increase in RR during heat stress preceded T(core) by 1 d for both groups. During cyclic heat, T(core) decreased by approximately 0.4% compared with constant heat in both the control and bST-treated groups. Bovine somatotropin treatment increased milk production similarly during the Tn and heat-stress periods, approximately 8.3% over the control; however, the bST-induced increase in milk-to-feed ratio was greatest during the continuous and cyclic heat-stress phases, approximately 16.2%. This increase occurred together with the elevation in T(core).

Genomic analysis of the impact of fescue toxicosis on hepatic function.

Fescue toxicosis is caused by consumption of toxins produced by an endophytic fungus, Neotyphodium coenophialum, in tall fescue [Lolium arundinaceum (Schreb.) Darbysh]. Microarray analysis was used to identify shifts in genetic expression associated with the affected physiological processes to identify potential targets for future pharmacological/toxicological intervention. Male rats (n = 24) were implanted with temperature transmitters, which measure core temperature every 5 min. After an 8-d recovery, the rats were fed an endophyte-free diet for 5 d. During the following 5-d treatment period, rats were fed either an endophyte-free or an endophyte-infected (91.5 microg of ergovaline.kg of BW(-1).d(-1)) diet. At the end of treatment, rats were euthanized and a sample of the liver was obtained. Feed conversion efficiency was calculated for both treatment groups. Serum prolactin concentrations were measured using ELISA. Liver tissue RNA was reverse transcribed and hybridized to an oligonucleotide microarray chip. Microarray data were analyzed using a 2-step ANOVA model and validated by quantitative real-time PCR. Significant reductions in mean core temperature, feed intake, feed conversion efficiency, BW, liver weight per unit of BW, and serum prolactin concentrations were observed in endophyte-infected rats. There was downregulation (P < 0.05) of various genes associated with energy metabolism, growth and development, and antioxidant protection, as well as an upregulation of genes associated with gluconeogenesis, detoxification, and biotransformation. This study demonstrated that even short-term exposure of rats to tall fescue endophytic toxins under thermoneutral conditions can result in physiological responses associated with altered gene expression within the liver.