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Mitochondrial dysfunction has been implicated in the adverse effects of nucleoside reverse transcriptase inhibitors (NRTIs) used to treat HIV-1 infections. To gain insight into the mechanism by which NRTIs alter mitochondrial function, the expression level of 542 genes associated with mitochondrial structure and functions was determined in the livers of p53 haplodeficient (+/-) C3B6F1 female mouse pups using mouse mitochondria-specific oligonucleotide microarray. The pups were transplacentally exposed to zidovudine (AZT) at 240 mg/kg bw/day or a combination of AZT and lamivudine (3TC) at 160 and 100mg/kg bw/day, respectively, from gestation day 12 through 18, followed by continuous treatment by oral administration from postnatal day 1-28. In addition, AZT/3TC effect was investigated in wild-type (+/+) C3B6F1 female mice. The genotype did not significantly affect the gene expression profile induced by AZT/3TC treatment. However, the transcriptional level of several genes associated with oxidative phosphorylation, mitochondrial tRNAs, fatty acid oxidation, steroid biosynthesis, and a few transport proteins were significantly altered in pups treated with AZT and AZT/3TC compared to their vehicle counterparts. Interestingly, AZT/3TC altered the expression level of 153 genes with false discovery rate of less than 0.05, in contrast to only 20 genes by AZT alone. These results suggest that NRTI-related effect on expression level of genes associated with mitochondrial functions was much greater in response to AZT/3TC combination treatment than AZT alone.

K-ras codon 12 GGT-->GAT and GGT-->GTT mutations are the most frequently observed K-ras point mutations in human and rodent tumors and therefore are implicated in carcinogenesis for many tissues. Measurement of these mutations in rat models and human tissue could facilitate a more logical extrapolation of rodent tumorigenesis data to human disease. We have developed allele-specific competitive blocker PCR (ACB-PCR) assays for rat K-ras codon 12 GGT-->GTT and GGT-->GAT mutations that parallel the already published assays for human K-ras codon 12 mutations. Liver K-ras codon 12 mutant allele fractions were measured in vehicle-treated and N-hydroxy-2-acetylaminofluorene (N-OH-AAF)-treated Big Blue rats. The average K-ras codon 12 GGT-->GTT mutant fraction (MF) for four control rats was 50 x 10(-6) (95% CI: 27 x 10(-6), 95 x 10(-6)) and for four treated rats was 165 x 10(-6) (95% CI: 87 x 10(-6), 312 x 10(-6)), indicating a 3.3-fold increase with treatment (95% CI: 1.3-8.1). The average MF of K-ras codon 12 GGT-->GAT for control rats was 1320 x 10(-6) (95% CI: 498 x 10(-6), 3500 x 10(-6)) and for treated rats was 8450 x 10(-6) (95% CI: 3180 x 10(-6), 22 400 x 10(-6)), indicating a 6.4-fold increase with treatment (95% CI: 1.6-25.4). These transgenic rats were part of a study that included analysis of liver lacI mutations. Although data from lacI determinations show that this compound induces mostly G-->T mutations, using the ACB-PCR method both K-ras codon 12 GGT-->GTT and GGT-->GAT MFs were significantly increased in treated rats versus control rats. This data raises the possibility that N-OH-AAF may not only induce mutations by a genotoxic mechanism, but also by amplification of both de novo and pre-existing K-ras mutation.

Background: Gender differences in gene expression were estimated in liver samples from 9 males and 9 females. The study tested 31,110 genes for a gender difference using a design that adjusted for sources of variation associated with cDNA arrays, normalization, hybridizations and processing conditions., Results: The genes were split into 2,800 that were clearly expressed (expressed genes) and 28,310 that had expression levels in the background range (not expressed genes). The distribution of p-values from the 'not expressed' group was consistent with no gender differences. The distribution of p-values from the 'expressed' group suggested that 8% of these genes differed by gender, but the estimated fold-changes (expression in males/expression in females) were small. The largest observed fold-change was 1.55. The 95% confidence bounds on the estimated fold-changes were less than 1.4 fold for 79.3%, and few (1.1%) exceed 2-fold., Conclusion: Observed gender differences in gene expression were small. When selecting genes with gender differences based upon their p-values, false discovery rates exceed 80% for any set of genes, essentially making it impossible to identify any specific genes with a gender difference.

The utility of liver H-ras codon 61 CAA to AAA mutant fraction as a biomarker of liver tumor development was investigated using neonatal male mice treated with 4-aminobiphenyl (4-ABP). Treatment with 0.1, 0.3, or 1.0 mumol 4-ABP produced dose-dependent increases in liver DNA adducts in B6C3F(1) and C57BL/6N mice. Eight months after treatment with 0.3 mumol 4-ABP or the DMSO vehicle, H-ras codon 61 CAA to AAA mutant fraction was measured in liver DNA samples (n = 12) by allele-specific competitive blocker-polymerase chain reaction (ACB-PCR). A significant increase in average mutant fraction was found in DNA of 4-ABP-treated mice, with an increase from 1.3 x 10(-5) (control) to 44.9 x 10(-5) (treated) in B6C3F(1) mice and from 1.4 x 10(-5) to 7.0 x 10(-5) in C57BL/6N mice. Compared with C57BL/6N mutant fractions, B6C3F(1) mutant fractions were more variable and included some particularly high mutant fractions, consistent with the more rapid development of liver foci expected in B6C3F(1) mouse liver. Twelve months after treatment, liver tumors developed in 79.2% of 4-ABP-treated and 22.2% of control B6C3F(1) mice; thus measurement of H-ras mutant fraction correlated with subsequent tumor development. This study demonstrates that ACB-PCR can directly measure background levels of somatic oncogene mutation and detect a carcinogen-induced increase in such mutation., (Published 2005 Wiley-Liss, Inc.)

Daily, rhythmic variation in various biochemical, physiological, and behavioral events is a fundamental property of biological organization. Here, we report analysis of relative levels of gene expression in the liver of 16 Fischer 344 rats as a function of time of day. Expression levels were determined for 3906 genes using high-density oligonucleotide microarrays. Of the 3906 genes, 1171 (30%) were clearly expressed while 2735 (70%) were not expressed or the expression was too low to distinguish from background levels. The maximum estimated changes observed for most genes (1029, 88%) were less than 1.5-fold. Analysis of variance and the Kruskal-Wallis tests were used to identify 67 genes whose expression was significantly altered as a function of time of day. These significantly altered genes were classified according to their functions and fall into key cellular pathways including drug metabolism, ion transport, signal transduction, DNA binding and regulation of transcription, and immune response.

NCTR measured sphinganine concentrations in the livers of mice and in the livers and kidneys of rats in conjunction with a tumour bioassay. In our model of the tumour incidence, target-tissue levels of sphinganine serve as a biomarker for a dose response of fumonisin B1 on cell death. Initially we questioned the utility of sphinganine levels in this role because they were highly variable when compared across time points. In spite of this concern, a conceptual framework and data are presented that support the use of sphinganine as a biomarker for a dose response of fumonisin B1 on cell death. This framework is reasonably consistent with observed sphinganine concentrations in the examined tissues, the literature on fumonisin's effects on sphingolipid synthesis, and our hypothesized mechanism through which fumonisin B1 increases age-specific tumour incidence.

A single genetic factor may affect the realization of several enzymes. To investigate the extent of pattern pleiotropy in the mouse, the activities of 28 enzymes in livers and brains from an inbred stock of C57BL/6JNctr and five F1 stocks heterozygous for known electrophoretic variants were measured. Five congenic backcross stocks of C57BL/6J, each homozygous for one or more electrophoretic markers, were mated with C57BL/6JNctr to construct the heterozygous variant F1 stocks. One of the five F1 stocks had no enzyme activities significantly different from those of C57BL/6JNctr, while two had one enzyme, one had four enzymes, and another had six enzymes with activities that were significantly different from those of C57BL/6JNctr. The latter two F1 stocks with multiple activity differences were those having the largest proportion of their genome of donor origin. Two of the F1 stocks were different from each other for one enzyme, and two were different for another enzyme. These differences and the relationship of these enzyme activities to the variant genes suggest that several genetic factors may affect an enzyme's realization.

The influence of epidermal growth factor (EGF), 0.75 micrograms g1; insulin, 1.5 micrograms g-1; glucagon, 1.25 micrograms g-1 and their combinations on the activities of hepatic pyruvate kinase (PK) and malic enzymes (ME) was monitored. Male CD2F1 mice were treated toward the end of the light or dark periods, 9 or 23 hours after lights on (9 or 23 HALO), and subgroups of six mice were killed at 4, 8 or 12 hr post-treatment. PK and ME activities from control mice were well characterized by cosine curves. The PK activity was maximal when ME activity was minimal at the transition from light to dark (9 HALO plus 4 hr) and PK was at a minimum when ME was highest (23 HALO plus 4 hr). Both enzymes were influenced by at least one peptide hormone, and the effects were strongly circadian-stage dependent. The only effect attributed to EGF was an increase of PK activity (23%) 12 hr after injection at 23 HALO. PK activity was increased by insulin (23%) at 23 HALO (4 hr after injection), but not at 9 HALO, and decreased (17%) by glucagon 12 hr after injection at 9 HALO. Several reductions in PK activity in response to various combinations of peptides were observed, and appeared to be caused by glucagon but influenced by insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Mice were standardized to 12 h of light exposure alternating with 12 h of darkness and were fed ad libitum. The mice in the first group were 8 weeks old and in the other 104-116 weeks old. Subgroups of 7 animals from each age group were killed at 6 circadian stages. Enzyme activities of 23 enzymes from liver or brain were measured by an analysis of variance for each age group. All but 1 enzyme from young mice, and all but 9 from older mice showed significant changes over time (p less than 0.01). The data from the 22 enzymes from young mice and the 14 enzymes from old mice were fit to the cosinor regression model to further characterize the rhythm. 15 enzymes from the young and 8 from the aged mice showed a significant regression to a 24-hour cosine curve (p less than 0.01); of the 8, 7 were the same enzymes in both groups. Amplitude changes, where they could be compared from the cosinor data (7 enzymes), were not statistically different. When total variance was compared, 12 enzymes showed unequal variance. Of these, old mice had the larger variance in 9 enzymes. Another difference between the young and the old was changes in mean enzyme activities. 12 enzymes from aged mice had decreased mesors, 2 had increased mesors, and 9 were unchanged. In general, our data suggest that some enzyme activity rhythms were lost, others were altered and a few were not affected by aging. In the case of many enzymes, older mice have a diminished ability to synchronize to the light signals.

The activities of 23 brain or liver enzymes were studied in 5-6 week old C57BL/6JNctr male and female mice that had been fed ad libitum and standardized for 2 weeks to either (1) 12 hr of light (0600-1800) alternating with 12 hr of darkness (1800-0600) (LD 12:12), (2) staggered sequences of 12 hr of light and 12 hr of dark (SLD 12:12) or (3) continuous illumination (LL 12:12) for 2 weeks. Mice in the LD 12:12 and LL 12:12 experiments were killed at 4 hr intervals along a 24-hr span in order to sample at six different circadian stages. Lighting schedules for mice in the SLD 12:12 experiment were organized such that six different circadian stages were sampled when all mice were killed at one time of day. All 23 enzymes demonstrated a prominent circadian rhythm in at least one of the experiments. Moreover, about two-thirds of the enzymes in LD and SLD 12:12 had a statistically significant fit to a 24-hr cosine curve, while only one-third of the enzymes in LL 12:12 had significant fits to cosine curves. Peak activities of enzymes from mice in LD 12:12 were clustered at the time of transition from light to dark. This was also the trend for the activities of enzymes from mice in SLD 12:12, but resynchronization did not appear completed within the 2-week span. This, along with the observation that mesors (mean 24-hr activity) were reduced and amplitudes altered, indicated that the 2-week standardization period was not sufficient for some enzymes. Times of peak activities, mesors and amplitudes were affected for most enzymes from mice in the LL 12:12 environment. This suggests that individual mice became desynchronized from one another with respect to the original light-dark schedule and that rhythms were altered or lost because individual mice were free running with frequencies different from 24 hr.