The science of toxicology is devoted, in large part, to understanding mechanisms of toxicity so that we can more accurately assess the risk posed by exposure to xenobiotic agents and, perhaps, ...intervene in the toxicologic process to mitigate harm. Dioxin-like chemicals continue to be of great concern as environmental toxicants. About 30 years ago the aryl hydrocarbon receptor (AHR) was discovered as a specific binding site for 2,3,7,8-tetrachlorodibenzo-p-dioxin. This giant step led to our current view that essentially all toxic effects of dioxins are AHR-mediated. The AHR serves as the archetype for understanding toxicity mediated by other soluble receptors. The fact that toxicity is receptor-mediated has important implications, especially for dose–response relationships. In laboratory animals genetic differences in AHR gene structure lead to profound differences in responsiveness to dioxin-like chemicals. Humans, however, exhibit relatively few AHR polymorphisms and these seem to exert only modest effects on downstream events. Dioxin toxicity is fundamentally due to AHR-mediated dysregulation of gene expression. Our current challenging goal is to determine which dysregulated genes underlie specific forms of dioxin toxicity. Mapping AHR-mediated gene expression in a variety of biological systems may help explain why dramatic differences in susceptibility to dioxin toxicity exist among laboratory species and why humans appear to be relatively resistant to adverse effects of dioxins.
The mammalian CYP1A1 , CYP1A2 , and CYP1B1 genes (encoding cytochromes P450 1A1, 1A2, and 1B1, respectively) are regulated by the aromatic hydrocarbon receptor (AHR).
The CYP1 enzymes are responsible ...for both metabolically activating and detoxifying numerous polycyclic aromatic hydrocarbons
(PAHs) and aromatic amines present in combustion products. Many substrates for CYP1 enzymes are AHR ligands. Differences in
AHR affinity between inbred mouse strains reflect variations in CYP1 inducibility and clearly have been shown to be associated
with differences in risk of toxicity or cancer caused by PAHs and arylamines. Variability in the human AHR affinity exists,
but differences in human risk of toxicity or cancer related to AHR activation remain unproven. Mouse lines having one or another
of the Cyp1 genes disrupted have shown paradoxical effects; in the test tube or in cell culture these enzymes show metabolic activation
of PAHs or arylamines, whereas in the intact animal these enzymes are sometimes more important in the role of detoxification
than metabolic potentiation. Intact animal data contradict pharmaceutical company policies that routinely test drugs under
development; if a candidate drug shows CYP1 inducibility, further testing is generally discontinued for fear of possible toxic
or carcinogenic effects. In the future, use of âhumanizedâ mouse lines, containing a human AHR or CYP1 allele in place of the orthologous mouse gene, is one likely approach to show that the AHR and the CYP1 enzymes in human
behave similarly to that in mouse.
Conventional biochemical and molecular techniques identified previously several genes whose expression is regulated by the
aryl hydrocarbon receptor (AHR). We sought to map the complete spectrum of ...AHR-dependent genes in male adult liver using expression
arrays to contrast mRNA profiles in Ahr -null mice ( Ahr â/â ) with those in mice with wild-type AHR ( Ahr + / + ). Transcript profiles were determined both in untreated mice and in mice treated 19 h earlier with 1000 μg/kg 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD). Expression of 456 ProbeSets was significantly altered by TCDD in an AHR-dependent manner, including members
of the classic AHRE-I gene battery, such as Cyp1a1 , Cyp1a2 , Cyp1b1 , and Nqo1. In the absence of exogenous ligand, AHR status alone affected expression of 392 ProbeSets, suggesting that the
AHR has multiple functions in normal physiology. In Ahr â/â mice, only 32 ProbeSets exhibited responses to TCDD, indicating that the AHR is required for virtually all transcriptional
responses to dioxin exposure in liver. The flavin-containing monooxygenases, Fmo2 and Fmo3, considered previously to be uninducible,
were highly induced by TCDD in an AHR-dependent manner. The estrogen receptor α as well as two estrogen-receptor-related genes
(α and γ) exhibit AHR-dependent expression, thereby extending cross-talk opportunities between the intensively studied AHR
and estrogen receptor pathways. p53 binding sites are over-represented in genes down-regulated by TCDD, suggesting that TCDD
inhibits p53 transcriptional activity. Overall, our study identifies a wide range of genes that depend on the AHR, either
for constitutive expression or for response to TCDD.
Colorectal cancer literature regarding the interaction between polymorphisms in carcinogen-metabolizing enzymes and red meat
intake/doneness is inconsistent. A case-control study was conducted to ...evaluate the interaction between red meat consumption,
doneness, and polymorphisms in carcinogen-metabolizing enzymes. Colorectal cancer cases diagnosed 1997 to 2000, ages 20 to
74 years, were identified through the population-based Ontario Cancer Registry and recruited by the Ontario Family Colorectal
Cancer Registry. Controls were sex-matched and age group-matched random sample of Ontario population. Epidemiologic and food
questionnaires were completed by 1,095 cases and 1,890 controls; blood was provided by 842 and 1,251, respectively. Multivariate
logistic regression was used to obtain adjusted odds ratio (OR) estimates. Increased red meat intake was associated with increased
colorectal cancer risk OR (>5 versus ≤2 servings/wk), 1.67 (1.36-2.05). Colorectal cancer risk also increased significantly
with well-done meat intake OR (>2 servings/wk well-done versus ≤2 servings/wk rare-regular), 1.57 (1.27-1.93). We evaluated
interactions between genetic variants in 15 enzymes involved in the metabolism of carcinogens in overcooked meat (cytochrome
P 450, glutathione S -transferase, UDP-glucuronosyltransferases, SULT, NAT, mEH, and AHR). CYP2C9 and NAT2 variants were associated with colorectal
cancer risk. Red meat intake was associated with increased colorectal cancer risk regardless of genotypes; however, CYP1B1
combined variant and SULT1A1-638G>A variant significantly modified the association between red meat doneness intake and colorectal
cancer risk. In conclusion, well-done red meat intake was associated with an increased risk of colorectal cancer regardless
of carcinogen-metabolizing genotype, although our data suggest that persons with CYP1B1 and SULT1A1 variants had the highest
colorectal cancer risk. (Cancer Epidemiol Biomarkers Prev 2008;17(11):3098–107)
The major toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) appear to result from dysregulation of mRNA levels mediated by the aryl hydrocarbon receptor (AHR). Dioxin-like chemicals alter ...expression of numerous genes in liver, but it remains unknown which lie in pathways leading to major toxicities such as hepatotoxicity, wasting and lethality. To identify genes involved in these responses we exploited a rat genetic model. Rats expressing an AHR splice-variant lacking a portion of the transactivation domain are highly resistant to dioxin-induced toxicities. We examined changes in hepatic mRNA abundances 19 hours after TCDD treatment in two dioxin-resistant rat strains/lines and two dioxin-sensitive rat strains/lines.
Resistant rat strains/lines exhibited fewer transcriptional changes in response to TCDD than did rats with wildtype AHR. However, well-known AHR-regulated and dioxin-inducible genes such as CYP1A1, CYP1A2, and CYP1B1 remained fully responsive to TCDD in all strains/lines. Pathway analysis indicated that the genes which respond differently to TCDD between sensitive and resistant rats are mainly involved in lipid metabolism, cellular membrane function and energy metabolism. These pathways previously have been shown to respond differently to dioxin treatment in dioxin-sensitive versus dioxin-resistant rats at a biochemical level and in the differential phenotype of toxicologic responses.
The transactivation-domain deletion in dioxin-resistant rats does not abolish global AHR transactivational activity but selectively interferes with expression of subsets of genes that are candidates to mediate or protect from major dioxin toxicities such as hepatotoxicity, wasting and death.
Mouse and rat models are mainstays in pharmacology, toxicology and drug development -- but differences between strains and between species complicate data interpretation and application to human ...health. Dioxin-like polyhalogenated aromatic hydrocarbons represent a major class of environmentally and economically relevant toxicants. In mammals dioxin exposure leads to a broad spectrum of adverse affects, including hepatotoxicity of varying severity. Several studies have shown that dioxins extensively alter hepatic mRNA levels. Surprisingly, though, analysis of a limited portion of the transcriptome revealed that rat and mouse responses diverge greatly (Boverhof et al. Toxicol Sci 94:398-416, 2006).
We employed oligonucleotide arrays to compare the response of 8,125 rat and mouse orthologs. We confirmed that there is limited inter-species overlap in dioxin-responsive genes. Rat-specific and mouse-specific genes are enriched for specific functional groups which differ between species, conceivably accounting for species-specificities in liver histopathology. While no evidence for the involvement of copy-number variation was found, extensive inter-species variation in the transcriptional-regulatory network was identified; Nr2f1 and Fos emerged as candidates to explain species-specific and species-independent responses, respectively.
Our results suggest that a small core of genes is responsible for mediating the similar features of dioxin hepatotoxicity in rats and mice but non-overlapping pathways are simultaneously at play to result in distinctive histopathological outcomes. The extreme divergence between mouse and rat transcriptomic responses appears to reflect divergent transcriptional-regulatory networks. Taken together, these data suggest that both rat and mouse models should be used to screen the acute hepatotoxic effects of drugs and toxic compounds.
Profiling of mRNA abundances with high-throughput platforms such as microarrays and RNA-seq has become an important tool in both basic and biomedical research. However, these platforms remain prone ...to systematic errors and have challenges in clinical and industrial applications. As a result, it is standard practice to validate a subset of key results using alternate technologies. Similarly, clinical and industrial applications typically involve transitions from a high-throughput discovery platform to medium-throughput validation ones. These medium-throughput validation platforms have high technical reproducibility and reduced sample input needs, and low sensitivity to sample quality (e.g., for processing FFPE specimens). Unfortunately, while medium-throughput platforms have proliferated, there are no comprehensive comparisons of them. Here we fill that gap by comparing two key medium-throughput platforms--NanoString's nCounter Analysis System and ABI's OpenArray System--to gold-standard quantitative real-time RT-PCR. We quantified 38 genes and positive and negative controls in 165 samples. Signal:noise ratios, correlations, dynamic range, and detection accuracy were compared across platforms. All three measurement technologies showed good concordance, but with divergent price/time/sensitivity trade-offs. This study provides the first detailed comparison of medium-throughput RNA quantification platforms and provides a template and a standard data set for the evaluation of additional technologies.
The aryl hydrocarbon receptor (AHR) participates in a wide range of critical cellular events in response to endogenous signals or xenobiotic chemicals. Hence, it is important that AHR levels and ...activity themselves be well controlled in target tissues. The AHR is essentially ubiquitous in its distribution in mammalian tissues. However, levels of the receptor vary widely across different tissues and among different cell types. AHR levels and activity are modulated by exposure to the receptor's own ligands and are influenced by other xenobiotic chemicals. Many different factors impinge on AHR levels and AHR activity. These factors may alter responsiveness of downstream pathways, thereby affecting normal physiologic functions as well as responses to toxic environmental chemicals such as dioxins. Our commentary appraises the current literature on factors that regulate AHR levels/activity and attempts to identify fruitful strategies towards discovery of key pathways by which AHR levels are modulated in response to endogenous signals and in response to xenobiotic chemicals. An extraordinarily large number of agents alter the level or activity of the AHR. We have not yet entered an age of enlightenment sufficient to achieve true understanding of the interplay of mechanisms that regulate AHR expression in space and in time.
Clustering has become an integral part of microarray data analysis and interpretation. The algorithmic basis of clustering -- the application of unsupervised machine-learning techniques to identify ...the patterns inherent in a data set -- is well established. This review discusses the biological motivations for and applications of these techniques to integrating gene expression data with other biological information, such as functional annotation, promoter data and proteomic data.
Despite several decades of research, the complete mechanism by which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other xenobiotic agonists of the aryl hydrocarbon receptor (AHR) cause toxicity ...remains unclear. While it has been shown that the AHR is required for all major manifestations of toxicity, the specific downstream changes involved in the development of toxic phenotypes remain unknown. Here we examine a panel of 13 genes that are AHR-regulated in many species and tissues. We profiled their hepatic mRNA abundances in two rat strains with very different sensitivities to TCDD: the TCDD-sensitive Long–Evans (Turku/AB; L–E) and the TCDD-resistant Han/Wistar (Kuopio; H/W). We evaluated doses ranging from 0 to 3000μg/kg at 19h after TCDD exposure and time points ranging from 1.5 to 384h after exposure to 100μg/kg TCDD. Twelve of 13 genes responded to TCDD in at least one strain, and seven of these showed statistically significant inter-strain differences in the time course analysis (Aldh3a1, Cyp1a2, Cyp1b1, Cyp2a1, Fmo1, Nfe2l2 and Nqo1). Cyp2s1 did not respond to TCDD in either rat strain. Five genes exhibited biphasic responses to TCDD insult (Ahrr, Aldh3a1, Cyp1b1, Nfe2l2 and Nqo1), suggesting a secondary event, such as association with additional transcriptional modulators. Of the 12 genes that responded to TCDD during the dose–response analysis, none had an ED50 equivalent to that of Cyp1a1, the most sensitive gene in this study, while nine genes responded to doses at least 10–100 fold higher, in at least one strain (Ahrr (L–E), Aldh3a1 (both), Cyp1a2 (both), Cyp1b1 (both), Cyp2a1 (L–E), Inmt (both), Nfe2l2 (L–E), Nqo1 (L–E) and Tiparp (both)). These data shed new light on the association of the AHR target genes with TCDD toxicity, and in particular the seven genes exhibiting strain-specific differences represent strong candidate mediators of Type-II toxicities.
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•NanoString measured hepatic mRNA molecules following TCDD treatment.•TCDD-sensitive Long–Evans and TCDD-resistant Han/Wistar rats were compared.•Time courses and dose responses were analyzed for AHR-core gene changes.•7 genes displayed inter-strain mRNA differences at times after TCDD exposure.•2 of the AHR-core genes had significant inter-strain differences in their TCDD ED50.
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