Genetic analysis of complex traits in the emerging Collaborative Cross.

The Collaborative Cross (CC) is a mouse recombinant inbred strain panel that is being developed as a resource for mammalian systems genetics. Here we describe an experiment that uses partially inbred CC lines to evaluate the genetic properties and utility of this emerging resource. Genome-wide analysis of the incipient strains reveals high genetic diversity, balanced allele frequencies, and dense, evenly distributed recombination sites-all ideal qualities for a systems genetics resource. We map discrete, complex, and biomolecular traits and contrast two quantitative trait locus (QTL) mapping approaches. Analysis based on inferred haplotypes improves power, reduces false discovery, and provides information to identify and prioritize candidate genes that is unique to multifounder crosses like the CC. The number of expression QTLs discovered here exceeds all previous efforts at eQTL mapping in mice, and we map local eQTL at 1-Mb resolution. We demonstrate that the genetic diversity of the CC, which derives from random mixing of eight founder strains, results in high phenotypic diversity and enhances our ability to map causative loci underlying complex disease-related traits.

Induction of oxidative stress and DNA damage in rat brain by a folate/methyl-deficient diet.

The age-associated decline in cellular antioxidant defenses and resultant accumulation of DNA damage in central nervous system has been mechanistically implicated in the etiology and pathogenesis of neurodegenerative diseases. Neurons possess a high metabolic activity and are especially vulnerable to the long-term effects of continuous exposure to endogenous reactive oxygen species. It is well recognized that adequate availability of essential nutrients involved in cellular one-carbon metabolism is essential for normal brain development and function. Additionally, the synthesis of the primary low-molecular cellular antioxidant glutathione is inter-dependently linked to one-carbon metabolic pathway. Thus, any aberrant disruptions in one-carbon metabolism can result in potentially deleterious effects including cell death as a result of an imbalance in the cellular redox state. Hence, in the present study, we examined the long-term effects of a folate/methyl-deficient (FMD) diet on cellular antioxidant defenses and DNA damage in the rat brain. Feeding male Fisher 344 rats a FMD diet resulted in perturbations in the levels of one-carbon metabolites along with induction of oxidative stress and oxidative DNA damage in the brain. This was evidenced by a decrease in the reduced oxidized/glutathione ratio, imbalance of cellular antioxidant defense system; specifically, altered activity and expression of antioxidant enzymes Mn-containing superoxide dismutase (Mn-SOD), catalase, and glutathione peroxidase (GPX), increased accumulation of oxidative DNA lesions, 8-hydroxydeoxyguanosine (8-OH-dG) and DNA single-strand breaks, even in the presence of increased expression of critical DNA repair genes apurinic/apyrimidinic endonuclease 1 (Apex1) and DNA polymerase beta (Polbeta), and apoptosis in the brains of folate/methyl-deficient rats. These results indicate that chronic methyl group deficiency leads to an imbalance in cellular antioxidant defense systems, increased oxidative stress, and apoptosis. Any of these events may compromise normal central nervous system function and contribute to the development of various neurological, behavioral, and neurocognitive dysfunctions.

Multicenter study of acetaminophen hepatotoxicity reveals the importance of biological endpoints in genomic analyses.

Gene expression profiling is a widely used technique with data from the majority of published microarray studies being publicly available. These data are being used for meta-analyses and in silico discovery; however, the comparability of toxicogenomic data generated in multiple laboratories has not been critically evaluated. Using the power of prospective multilaboratory investigations, seven centers individually conducted a common toxicogenomics experiment designed to advance understanding of molecular pathways perturbed in liver by an acute toxic dose of N-acetyl-p-aminophenol (APAP) and to uncover reproducible genomic signatures of APAP-induced toxicity. The nonhepatotoxic APAP isomer N-acetyl-m-aminophenol was used to identify gene expression changes unique to APAP. Our data show that c-Myc is induced by APAP and that c-Myc-centered interactomes are the most significant networks of proteins associated with liver injury. Furthermore, sources of error and data variability among Centers and methods to accommodate this variability were identified by coupling gene expression with extensive toxicological evaluation of the toxic responses. We show that phenotypic anchoring of gene expression data is required for biologically meaningful analysis of toxicogenomic experiments.

Phenotypic anchoring of acetaminophen-induced oxidative stress with gene expression profiles in rat liver.

Toxicogenomics provides the ability to examine in greater detail the underlying molecular events that precede and accompany toxicity, thus allowing prediction of adverse events at much earlier times compared to classical toxicological end points. Acetaminophen (APAP) is a pharmaceutical that has similar metabolic and toxic responses in rodents and humans. Recent gene expression profiling studies with APAP found an oxidative stress signature at a subtoxic dose that we hypothesized can be phenotypically anchored to conventional biomarkers of oxidative stress. Liver tissue was obtained from experimental animals used to generate microarray data, where male rats were given APAP at subtoxic (150 mg/kg) or overtly toxic (1500 and 2000 mg/kg) doses and sacrificed at 6, 24, or 48 h. Oxidative stress in liver was evaluated by a diverse panel of markers that included assessing expression of base excision repair (BER) genes, quantifying oxidative lesions in genomic DNA, and evaluating protein and lipid oxidation. A subtoxic dose of APAP produced significant accumulation of nitrotyrosine protein adducts. Both subtoxic and toxic doses caused a significant increase in 8-hydroxy-deoxyguanosine (8-OH-dG) as well as a significant decrease in glutathione (GSH) content. Only toxic doses of APAP significantly induced expression levels of BER genes. None of the doses examined resulted in a significant increase in the number of abasic sites or in the amount of lipid peroxidation. The accumulation of nitrotyrosine and 8-OH-dG adducts along with reduced GSH content in the liver phenotypically anchors the oxidative stress gene expression signature observed with a subtoxic dose of APAP, lending support to the validity of gene expression studies as a sensitive and biologically meaningful end point in toxicology.

Expression of base excision DNA repair genes as a biomarker of oxidative DNA damage.

Oxidative stress induced DNA damage is considered to be the most common insult affecting the genome. Moreover, it is recognized as a common pathway to mutations and is suggested to play a major role in the development of chronic diseases such as cancer. However, current analytical methods used to detect oxidative DNA damage have been hampered by both technical and biological obstacles. These include spurious oxidation during DNA isolation and processing, and the inherent removal of damaged bases by numerous operating DNA repair systems. The removal of oxidized bases is performed predominantly by the base excision repair (BER) pathway and it has been shown that induction of DNA repair genes occurs in response to oxidative stress. Here, we demonstrate the utility of measuring changes in expression of BER genes as a sensitive in vivo biomarker for oxidative DNA damage.

Lipids versus proteins as major targets of pro-oxidant, direct-acting hemolytic agents.

Lipid peroxidation and the accompanying translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the lipid bilayer have recently been identified as key components of a signaling pathway for phagocytosis of apoptotic cells by macrophages. Drug-induced hemolytic anemia has long been known to be caused by an accelerated uptake of damaged (but intact) erythrocytes by macrophages in the spleen, and this process has been associated with enhanced formation of reactive oxygen species (ROS). However, the role of lipid peroxidation in hemolytic injury has remained unclear, and the effect of hemolytic agents on the distribution of PS in the erythrocyte membrane is unknown. The present studies were undertaken to determine whether lipid peroxidation and PS translocation could be detected in rat and human erythrocytes by three types of direct-acting hemolytic agents--dapsone hydroxylamine, divicine hydroquinone, and phenylhydrazine. 2',7'-Dichlorodihydrofluorescein diacetate was employed as a probe for intracellular ROS formation; lipid peroxidation was assessed by GC/MS analysis of F2-isoprostanes; and PS externalization was measured by annexin V labeling and the prothrombinase assay. The data confirmed that all three hemolytic agents generate ROS within erythrocytes under hemolytic conditions; however, no evidence for lipid peroxidation or PS translocation was detected. Instead, ROS production by these hemolytic agents was associated with extensive binding of oxidized and denatured hemoglobin to the membrane cytoskeleton. The data suggest that the transmembrane signal for macrophage recognition of hemolytic injury may be derived from oxidative alterations to erythrocyte proteins rather than to membrane lipids.

GTX(4) imposters: characterization of fluorescent compounds synthesized by Pseudomonas stutzeri SF/PS and Pseudomonas/Alteromonas PTB-1, symbionts of saxitoxin-producing Alexandrium spp.

Saxitoxins, the etiological agent of paralytic shellfish poisoning, are synthesized by dinoflagellates and cyanobacteria. Several reports indicate that bacteria are capable of saxitoxin synthesis. Two bacterial strains were isolated from saxitoxin-producing dinoflagellates, Alexandrium tamarense and A. lusitanicum (=Alexandrium minutum), and grown under a variety of culture conditions including those previously reported to induce saxitoxin synthesis in bacteria. Five fluorescent compounds were accumulated by the bacteria that had HPLC-FLD retention times similar to a reference standard of GTX(4), one of the saxitoxin congeners. However, we were unable to detect GTX(1), the epimeric partner of GTX(4), in the bacterial samples. The GTX(4) standard was hydrolyzed by NaOH/heat treatment but four of the bacterial compounds were stable. Unlike GTX(4), none of the five bacterial compounds were detectable by HPLC-FLD following electrochemical oxidation. The fluorescence emission spectrum of each of the five bacterial compounds was unique and readily discernable from the spectrum of GTX(4). None of the samples containing the putative GTX(4) toxin yielded positive results when analyzed by a 3H-saxitoxin receptor-binding assay for saxitoxin-like activity. We cannot rule out the possibility that these bacteria produce saxitoxins, however, our data clearly demonstrate that they accumulate at least five different fluorescent compounds that could be easily mistaken for GTX(4). We conclude that these five fluorescent compounds are GTX(4) imposters and that fluorescence scanning and chemical/heat stability should, at a minimum, be incorporated into HPLC-FLD protocols for identification of saxitoxins.

Development of a protocol for determination of domoic acid in the sand crab (Emerita analoga): a possible new indicator species.

The aim of this study was to begin evaluating the utility of sand crabs (Emerita analoga) as an indicator species for the algal neurotoxin, domoic acid (DA), in Monterey Bay, California, USA, a site of recurrent blooms of the DA-producing diatom, Pseudo-nitzschia. One of the current sentinel organisms, the sea mussel (Mytilus californianus), has shown minimal or undetectable toxicity during some local bloom events. As a critical step in assuring the accuracy of DA determinations in E. analoga, we have developed and validated a highly efficient extraction protocol that yields toxin recoveries of 97+/-2.9%. We also determined by HPLC-UV and receptor binding assay, with confirmation by LC-MS/MS, that sand crabs accumulated measurable amounts of DA during toxic Pseudo-nitzschia blooms, while the sea mussel showed no detectable toxin. In addition, a comparison of inter-animal variability in DA content revealed values ranging from ca. 0.5 to 5 microg DAg(-1) tissue and no consistent trend with size class, based on either animal weight or length. These data on the toxicity of individual animals will be useful in designing an appropriate sampling strategy for monitoring DA and, importantly, indicate that sand crabs do not appear to progressively bioaccumulate DA with age.

Receptor binding assay for paralytic shellfish poisoning toxins: optimization and interlaboratory comparison.

A receptor binding assay (RBA) for detection of paralytic shellfish poisoning (PSP) toxins was formatted for use in a high throughput detection system using microplate scintillation counting. The RBA technology was transferred from the National Ocean Service, which uses a Wallac TriLux 1450 MicroBeta microplate scintillation counter, to the California Department of Health Services, which uses a Packard TopCount scintillation counter. Due to differences in the detector arrangement between these 2 counters, markedly different counting efficiencies were exhibited, requiring optimization of the RBA protocol for the TopCount instrument. Precision, accuracy, and sensitivity [limit of detection = 0.2 microg saxitoxin (STX) equiv/100 g shellfish tissue] of the modified protocol were equivalent to those of the original protocol. The RBA robustness and adaptability were demonstrated by an interlaboratory study, in which STX concentrations in shellfish generated by the TopCount were consistent with MicroBeta-derived values. Comparison of STX reference standards obtained from the U.S. Food and Drug Administration and the National Research Council, Canada, showed no observable differences. This study confirms the RBA's value as a rapid, high throughput screen prior to testing by the conventional mouse bioassay (MBA) and its suitability for providing an early warning of increasing PSP toxicity when toxin levels are below the MBA limit of detection.

Mechanism for prevention of alcohol-induced liver injury by dietary methyl donors.

Alcohol-induced liver injury (ALI) has been associated with, among other molecular changes, abnormal hepatic methionine metabolism, resulting in decreased levels of S-adenosylmethionine (SAM). Dietary methyl donor supplements such as SAM and betaine mitigate ALI in animal models; however, the mechanisms of protection remain elusive. It has been suggested that methyl donors may act via attenuation of alcohol-induced oxidative stress. We hypothesized that the protective action of methyl donors is mediated by an effect on the oxidative metabolism of alcohol in the liver. Male C57BL/6J mice were administered a control high-fat diet or diet enriched in methyl donors with or without alcohol for 4 weeks using the enteral alcohol feeding model. As expected, attenuation of ALI and an increase in reduced glutathione:oxidized glutathione ratio were achieved with methyl donor supplementation. Interestingly, methyl donors led to a 35% increase in blood alcohol elimination rate, and while there was no effect on alcohol metabolism in the stomach, a profound effect on liver alcohol metabolism was observed. The catalase-dependent pathway of alcohol metabolism was induced, yet the increase in CYP2E1 activity by alcohol was blunted, which may be mitigating production of oxidants. Additional factors contributing to the protective effects of methyl donors in ALI were increased activity of low- and high-K(m) aldehyde dehydrogenases leading to lower hepatic acetaldehyde, maintenance of the efficient mitochondrial energy metabolism, and promotion of peroxisomal beta-oxidation. Profound changes in alcohol metabolism represent additional important mechanism of the protective effect of methyl donors in ALI.

Temporal correlation of pathology and DNA damage with gene expression in a choline-deficient model of rat liver injury.

Hepatocellular carcinoma (HCC) is the terminal event in chronic liver diseases with repeated cycles of cellular injury and regeneration. Although much is known about the cellular pathogenesis and etiological agents leading to HCC, the molecular events are not well understood. The choline-deficient (CD) model of rodent HCC involves the consecutive emergence of a fatty liver, apoptosis, compensatory proliferation, fibrosis, and cirrhosis that is markedly similar to the sequence of events typified by human HCC. Moreover, oxidative stress is thought to play a pivotal role in the progression of the disease. Here, we hypothesize that gene expression profiling can temporally mirror the histopathology and oxidative DNA damage observed with this model. We show that clusters of highly co-regulated genes representing distinct cellular pathways for lipid biosynthesis and metabolism, apoptosis, cell proliferation, and tissue remodeling temporally correlate with the well-defined sequential emergence of pathological alterations in the progression of liver disease. Additionally, an oxidative stress signature was observed that was corroborated in a time-dependent manner with increases in oxidized purines and abasic sites in DNA. Collectively, expression patterns were strongly driven by pathology, demonstrating that patterns of gene expression in advanced stages of liver disease are primarily driven by histopathological changes and to a much lesser degree by the original etiological agent. In conclusion, gene expression profiling coupled with the CD model of HCC provides a unique opportunity to unveil the molecular events associated with various stages of liver injury and carcinogenesis and to distinguish between causal and consecutive changes.