Changes in Faecal Microbiota Profile and Plasma Biomarkers following the Administration of an Antioxidant Oleuropein-Rich Leaf Extract in a Rat Model Mimicking Colorectal Cancer.

Oleuropein (OLE), a phenolic compound particularly abundant in the olive leaves, has been reported to have beneficial activities against colorectal cancer (CRC). In vitro studies suggested that these latter could be due to a modulation of the intestinal microbiota. Aiming to evaluate if OLE could affect the intestinal microbiota and the plasma metabolome, an antioxidant oleuropein-rich leaf extract (ORLE) was administered for one week to PIRC rats (F344/NTac-Apcam1137), a genetic model mimicking CRC. ORLE treatment significantly modulated the gut microbiota composition. Plasma metabolomic profiles revealed a significant predictive ability for amino acids, medium-chain fatty acids, and aldehydes. Pathway analysis revealed a significant decrease in phosphatidylcholine accumulation (LogFC = -1.67) in PIRC rats. These results suggest a significant effect of ORLE administration on faecal microbiota profiles and plasma metabolomes, thereby offering new omics-based insights into its protective role in CRC progression.

Effects of sodium nitrite reduction, removal or replacement on cured and cooked meat for microbiological growth, food safety, colon ecosystem, and colorectal carcinogenesis in Fischer 344 rats.

Epidemiological and experimental evidence indicated that processed meat consumption is associated with colorectal cancer risks. Several studies suggest the involvement of nitrite or nitrate additives via N-nitroso-compound formation (NOCs). Compared to the reference level (120 mg/kg of ham), sodium nitrite removal and reduction (90 mg/kg) similarly decreased preneoplastic lesions in F344 rats, but only reduction had an inhibitory effect on Listeria monocytogenes growth comparable to that obtained using the reference nitrite level and an effective lipid peroxidation control. Among the three nitrite salt alternatives tested, none of them led to a significant gain when compared to the reference level: vegetable stock, due to nitrate presence, was very similar to this reference nitrite level, yeast extract induced a strong luminal peroxidation and no decrease in preneoplastic lesions in rats despite the absence of NOCs, and polyphenol rich extract induced the clearest downward trend on preneoplastic lesions in rats but the concomitant presence of nitrosyl iron in feces. Except the vegetable stock, other alternatives were less efficient than sodium nitrite in reducing L. monocytogenes growth.

The Core Human Microbiome: Does It Exist and How Can We Find It? A Critical Review of the Concept.

The core microbiome, which refers to a set of consistent microbial features across populations, is of major interest in microbiome research and has been addressed by numerous studies. Understanding the core microbiome can help identify elements that lead to dysbiosis, and lead to treatments for microbiome-related health states. However, defining the core microbiome is a complex task at several levels. In this review, we consider the current state of core human microbiome research. We consider the knowledge that has been gained, the factors limiting our ability to achieve a reliable description of the core human microbiome, and the fields most likely to improve that ability. DNA sequencing technologies and the methods for analyzing metagenomics and amplicon data will most likely facilitate higher accuracy and resolution in describing the microbiome. However, more effort should be invested in characterizing the microbiome's interactions with its human host, including the immune system and nutrition. Other components of this holobiontic system should also be emphasized, such as fungi, protists, lower eukaryotes, viruses, and phages. Most importantly, a collaborative effort of experts in microbiology, nutrition, immunology, medicine, systems biology, bioinformatics, and machine learning is probably required to identify the traits of the core human microbiome.

Fish Consumption and Colorectal Cancer Risk: Meta-Analysis of Prospective Epidemiological Studies and Review of Evidence from Animal Studies.

Background: Epidemiological studies on the association between fish consumption and colorectal cancer (CRC) risk have yielded inconsistent results, despite evidence from preclinical studies that long-chain ω-3 polyunsaturated fatty acids inhibit colorectal carcinogenesis. We conducted a meta-analysis of prospective epidemiological studies investigating the association between fish consumption and CRC risk among humans and reviewed studies examining the link between fish components and colorectal carcinogenesis in animal models. Methods: We included studies published until November 2020. We calculated the summary risk ratio (SRR) and 95% confidence intervals (CI) through random effects meta-analysis models in order to summarize evidence from studies among humans. Results: Twenty-five prospective epidemiological studies encompassing 25,777 CRC cases were included. Individuals in the highest (vs. lowest) category of fish consumption had a significantly reduced risk of CRC (SRR 0.94, 95%CI 0.89-0.99). In dose-response meta-analysis, a 50-g increment in the daily consumption of fish was associated with a statistically significant 4% reduction in CRC risk (SRR 0.96, 95%CI 0.92-0.99). Preclinical studies (n = 25) identified multiple mechanisms of action of fish and fish components on colorectal carcinogenesis. Conclusions: Dietary recommendations for cancer prevention should take into account the evidence from epidemiological and preclinical studies that increasing fish consumption may be effective in preventing CRC.

Intestinal microbiota profiles in a genetic model of colon tumorigenesis correlates with colon cancer biomarkers.

Faecal (FM) and colon mucosal associated microbiota (MAM) were studied in a model of colorectal cancer (CRC), the Apc-mutated Pirc rats, and in age-paired wt F344 rats. Principal Coordinates Analysis indicated that samples' distribution was driven by age, with samples of young rats (1 month old; without tumours) separated from older ones (11-month-old; bearing tumours). Diversity analysis showed significant differences between FM and MAM in older Pirc rats, and between MAM of both Pirc and wt rats and the tumour microbiota, enriched in Enterococcus, Escherichia/Shigella, Proteus and Bifidobacteriaceae. In young animals, Pirc FM was enriched in the genus Delftia, while wt FM was enriched in Lactobacillus and Streptococcus. Some CRC biomarkers and faecal short chain fatty acids (SCFAs) were also measured. Colon proliferation and DClK1 expression, a pro-survival mucosal marker, were higher in Pirc than in wt rats, while the mucin MUC2, was lower in Pirc rats. Branched SCFAs were higher in Pirc than in wt animals. By Spearman analysis CRC biomarkers correlated with FM (in both young and old rats) and with MAM (in young rats), suggesting a specific relationship between the gut microbiota profile and these functional mucosal parameters deserving further investigation.

Colon fibroblasts from Pirc rats (F344/NTac-Apcam1137 ) exhibit a proliferative and inflammatory phenotype that could support early stages of colon carcinogenesis.

The role of fibroblast APC mutation in carcinogenesis is not clear. Apc+/- colon fibroblasts have been previously characterized: however, little is known about their behavior at very early-stage of colon carcinogenesis. We cultured colon mucosa fibroblasts (PCF, Apc+/- ) of Pirc rats (F344/NTac-Apcam1137 ) at an early stage of tumorigenesis, in absence of preneoplastic lesions, and of age-matched wt (WCF): DNA damage levels, inflammatory phenotype and the expression of known markers of CAFs were analyzed. The latter were also assessed by microarray analysis on colon normal mucosa of Pirc and wt animals. PCF exhibited higher proliferative rates (P < .001) and delayed replicative senescence onset (P < .05) compared to WCF, along with a lower level of oxidative DNA damage (P < .05). Furthermore, a constitutively higher expression of COX-2 and sensitivity to inflammatory stimuli was found in PCF compared to WCF (P < .05), accompanied by higher invasive capability (P < .05) and presence of cytoplasmic chromatin foci (cytoplasmic chromatin foci, P < .05). However, they neither expressed CAFs markers (α-SMA, IL-6) nor responded to CAFs activating stimuli (TGF-ß). Accordingly, CAFs markers and activating stimuli resulted down-regulated in Pirc normal mucosa compared to wt, whereas DNA damage response and tolerance pathways were overexpressed. These data show for the first time that a proliferative and inflammatory phenotype characterizes Apc+/- colon fibroblasts since very early stages of colon tumorigenesis, and indicate a role of Apc mutation in driving fibroblast phenotypic alterations that could support the establishment of a protumorigenic environment. Early pharmacological targeting of these dysfunctions might impact on tumor prevention in FAP patients.

Oleuropein-Rich Leaf Extract as a Broad Inhibitor of Tumour and Macrophage iNOS in an Apc Mutant Rat Model.

Oleuropein, the major compound found in olive leaves, has been reported to exert numerous pharmacological properties, including anti-inflammatory, anti-diabetic and anti-cancer effects. The purpose of this study was to evaluate, for the first time, the effect of oleuropein-rich leaf extracts (ORLE) in already-developed colon tumours arising in Apc (adenomatous polyposis coli) mutated PIRC rats (F344/NTac-Apcam1137). Here, we were able to investigate in parallel the anti-cancer effect of ORLE, both in vivo and in vitro, and its anti-inflammatory effect on macrophages, representing a critical and abundant population in most solid tumour microenvironment. We found that in vivo ORLE treatment promoted apoptosis and attenuated iNOS activity both in colon tumours as in peritoneal macrophages of PIRC rats. We this confirmed in vitro using primary RAW264.7 cells: ORLE reduced iNOS activity in parallel with COX-2 and pro-inflammatory cytokines, such as IL-1ß, IL-6 and TGF-ß. These findings suggest that ORLE possess a strong anti-inflammatory activity, which could be crucial for dampening the pro-tumourigenic activity elicited by a chronic inflammatory state generated by either tumour cells or tumour-associated macrophages.

DNA damage in colon mucosa of Pirc rats, an Apc-driven model of colon tumorigenesis.

APC mutation is the first event triggering colon carcinogenesis (CRC). The contribution of APC to colon mucosa DNA damage is not well characterized yet. Similarly, the role of genotoxin-producer gut microorganisms is unclear. DNA strand breaks and oxidative damage were measured in Pirc rats, mutated in Apc, with the COMET assay at age 1 (T1) and 11 months (T11), i.e. in absence and presence of colon adenomas. In Pirc colon mucosa a 2-fold increase in the mean level of DNA oxidative damage was found at T11 compared to T1. Moreover, the analysis of DNA damage distribution showed that the proportion of Pirc mucosa cells in the highest DNA damage class was increased compared to wt rats at T1 and T11 months (p < 0.05 and <0.001, respectively). The analysis of colon mucosa-associated microbiota composition showed that this result was not attributable to the presence of genotoxin-producer bacteria B. fragilis nor E. coli. However, Pirc colon mucosa was enriched in Clostridium cluster XI, harmful bacteria in the large intestine, while the wt colon mucosa was enriched in Clostridium cluster IV. This work provides an original way to investigate the interplay between Apc and gut microbiota in affecting DNA stability during CRC.

Cancer Glycolytic Dependence as a New Target of Olive Leaf Extract.

Oleuropein (Ole), the main bioactive phenolic component of Olea europaea L. has recently attracted the scientific attention for its several beneficial properties, including its anticancer effects. This study is intended to investigate whether an olive leaf extract enriched in Ole (OLEO) may counteract the aerobic glycolysis exploited by tumor cells. We found that OLEO decreased melanoma cell proliferation and motility. OLEO was also able to reduce the rate of glycolysis of human melanoma cells without affecting oxidative phosphorylation. This reduction was associated with a significant decrease of glucose transporter-1, protein kinase isoform M2 and monocarboxylate transporter-4 expression, possible drivers of such glycolysis inhibition. Extending the study to other tumor histotypes, we observed that the metabolic effects of OLEO are not confined to melanoma, but also confirmed in colon carcinoma, breast cancer and chronic myeloid leukemia. In conclusion, OLEO represents a natural product effective in reducing the glycolytic metabolism of different tumor types, revealing an extended metabolic inhibitory activity that may be well suited in a complementary anti-cancer therapy.

A flavonoid-rich extract from bergamot juice prevents carcinogenesis in a genetic model of colorectal cancer, the Pirc rat (F344/NTac-Apcam1137).

PURPOSE: To determine the potential of a flavonoid-rich extract from bergamot juice (BJe) to prevent colorectal carcinogenesis (CRC) in vivo. MAIN METHODS: Pirc rats (F344/NTac-Apcam1137), mutated in Apc, the key gene in CRC, were treated with two different doses of BJe (35 mg/kg or 70 mg/kg body weight, respectively) mixed in the diet for 12 weeks. Then, the entire intestine was surgically removed and dissected for histological, immunohistochemical and molecular analyses. RESULTS: Rats treated with BJe showed a significant dose-related reduction in the colon preneoplastic lesions mucin-depleted foci (MDF). Colon and small intestinal tumours were also significantly reduced in rats supplemented with 70 mg/kg of BJe. To elucidate the involved mechanisms, markers of inflammation and apoptosis were determined. Compared to controls, colon tumours from BJe 70 mg/kg-supplemented rats showed a significant down-regulation of inflammation-related genes (COX-2, iNOS, IL-1ß, IL-6 and IL-10 and Arginase 1). Moreover, in colon tumours from rats fed with 70 mg/kg BJe, apoptosis was significantly higher than in controls. Up-regulation of p53 and down-regulation of survivin and p21 genes was also observed. CONCLUSIONS: These data indicate a strong chemopreventive activity of BJe that, at least in part, is due to its pro-apoptotic and anti-inflammatory actions. This effect could be exploited as a strategy to prevent CRC in high-risk patients.

Supplementation with phytoestrogens and insoluble fibers reduces intestinal carcinogenesis and restores ER-ß expression in Apc-driven colorectal carcinogenesis.

Supplementation with phytoestrogens and insoluble fibers has been reported to reduce duodenal polyps in colectomized familial adenomatous polyposis patients, with a mechanism involving, at least in part, upregulation of estrogen receptor-ß subtype, whose expression is lowered during intestinal tumorigenesis. These data suggest a protective effect also in the colon, the main target organ for tumorigenesis in familial adenomatous polyposis and a major cancer type in non-familial (sporadic) cancers. Therefore, we tested whether a similar preparation might reduce tumorigenesis in the colon of Pirc rats (F344/NTac-Apc) mutated in the Apc gene and thus, like familial adenomatous polyposis patients, spontaneously developing multiple tumors in the colon. We first demonstrate that estrogen receptor-ß expression in Pirc rat colon is significantly down-regulated compared to age-matched wt rats. Then, Pirc rats aged 1 month were treated for 3 months with Adipol (Adi), a patented preparation containing phytoestrogens and insoluble fibers. Colon tumorigenesis was significantly reduced by Adi treatment (colon tumors/rat were 5.3 ± 0.8 and 2.9 ± 0.3, Mucin Depleted Foci/rat 127 ± 6.6 and 97.1 ± 8.6 in Controls and Adi-treated rats, respectively, means ± SE, P < 0.01). The treatment also normalized colon proliferation pattern along the crypt and significantly increased apoptosis in colon tumors. Estrogen receptor-ß expression was increased by Adi treatment, especially in the tumors. These positive effects suggest that Adipol may be exploited as a chemopreventive agent to reduce cancer risk in familial adenomatous polyposis patients and to postpone prophylactic colectomy. Moreover, given the similarities between familial adenomatous polyposis and sporadic colorectal cancer, it might also be used as chemopreventive agent in colorectal cancer patients at risk.

Fecal microbiome as determinant of the effect of diet on colorectal cancer risk: comparison of meat-based versus pesco-vegetarian diets (the MeaTIc study).

BACKGROUND: Convincing evidence suggests that the risk of colorectal cancer (CRC) is increased by the typical Western diet characterized by high consumption of red and processed meat. In addition, some epidemiological studies suggest a reduction in the risk of CRC associated with fish consumption. The role of the gut microbiome in this diet-associated risk is not well understood. METHODS/DESIGN: This is a randomized parallel open clinical trial comprising a total of 150 clinically healthy subjects randomly assigned to three groups: a meat-based diet of which 4 portions per week are red meat (1 portion = 150 g), 3 portions per week are processed meat (1 portion = 50 g), and 1 portion per week is poultry (1 portion = 150 g), for a total amount of 900 g per week of meat and derivatives; a meat-based diet supplemented with alpha-tocopherol; and a pesco-vegetarian diet excluding fresh and processed meat and poultry, but which includes 3 portions per week of fish for a total amount of 450 g per week. Each intervention will last 3 months. The three diets will be isocaloric and of three different sizes according to specific energy requirements. Anthropometric measurements, body composition, and blood and fecal samples will be obtained from each participant at the beginning and end of each intervention phase. The measure of the primary outcome will be the change from baseline in DNA damage induced by fecal water using the comet assay in a cellular model. Secondary outcome measures will be changes in the profile of fecal microbiomes, global fecal and urinary peroxidation markers, and neoplastic biomarkers. DISCUSSION: Although epidemiological data support the promoting role of meat and the possible protective role of fish in colon carcinogenesis, no study has directly compared dietary profiles characterized by the presence of these two food groups and the role of the gut microbiome in these diet-associated CRC risks. This study will test the effect of these dietary profiles on validated CRC risk biomarkers. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03416777. Registered on 3 May 2018.

High Sensitivity to Cholic Acid-induced Colonic Tumorigenesis Makes Female PIRC Rats (F344/NTac-Apcam1137) a Suitable Model for Studying CRC-promoting Agents.

BACKGROUND/AIM: Rats of the adenomatous polyposis coli (Apc)-mutated female polyposis in rat (PIRC) (F344/NTac-Apcam1137) model exhibit a low level of intestinal tumorigenesis and are thus potentially exploitable as a model for identifying substances increasing colorectal cancer (CRC). MATERIALS AND METHODS: To test this possibility, we treated such rats with the bile acid (BA) cholic acid (CA) (0.3% w/w in the diet), known to promote CRC, and assessed tumorigenesis. RESULTS: Precancerous colonic lesions (mucin-depleted foci) and intestinal tumors were dramatically increased in CA-treated rats compared to controls (p<0.01). Colon mucosa proliferation was higher and apoptosis lower than those in controls. Expression of nuclear receptor 1h4 (Nr1h4) gene [encoding for BA receptor farnesoid X receptor (FXR)], organic solute transporter beta (Ostb) and fatty acid-binding protein 6 (Fabp6), FXR-dependent BA transporters, were dramatically down-regulated in CA-treated rats. CONCLUSION: CA-increased tumorigenesis in female PIRC rats, with mechanisms involving increased proliferation, reduced apoptosis and marked down-regulation of genes controlling BA homeostasis. Since BAs have been implicated in CRC, we suggest that female PIRC rats can be used to identify CRC-promoting agents.

Morin-dependent inhibition of low molecular weight protein tyrosine phosphatase (LMW-PTP) restores sensitivity to apoptosis during colon carcinogenesis: Studies in vitro and in vivo, in an Apc-driven model of colon cancer.

LMW-PTP has been associated with the development of colorectal cancer (CRC) and with the resistance to chemotherapy in cancer cells. To clarify its role in vivo, we studied LMW-PTP expression in Pirc rats (F344/NTac-Apc am1137 ), genetically prone to CRC and resistant to apoptosis. In the morphologically normal mucosa (NM) of Pirc rats, a dramatic over-expression of LMW-PTP was found compared to wt rats (about 60 times higher). Moreover, LMW-PTP levels further increase in spontaneously developed Pirc colon tumors. To understand if and how LMW-PTP affects resistance to apoptosis, we studied CRC cell lines, sensitive (HT29 and HCT-116), or resistant (HT29R, HCT116R) to 5-Fluorouracil (5-FU): resistant cells over-express LMW-PTP. When resistant cells were challenged with morin, a polyphenol inhibiting LMW-PTP, a fast and dose-related down-regulation of LMW-PTP was observed. 5-FU and morin co-treatment dramatically decreased cell viability, increased apoptosis, and significantly impaired self-renewal ability of all the cancer cell lines we have studied. Similarly, we observed that, in Pirc rats, one-week morin administration (50 mg/kg) down-regulated LMW-PTP and restored the apoptotic response to 5-FU in the NM. Finally, administration of morin for a longer period led to a significant reduction in colon precancerous lesions, together with a down-regulation of LMW-PTP. Taken together, these results document the involvement of LMW-PTP in the process of CRC in vitro and in vivo. Morin treatment may be envisaged as a system to increase the sensitivity to chemotherapy and to prevent carcinogenesis.

Aging related changes in circulating reactive oxygen species (ROS) and protein carbonyls are indicative of liver oxidative injury.

Oxidative stress, defined as an imbalance between the production of reactive oxygen species (ROS) and antioxidant defense mechanisms, plays a major role in inducing oxidative damage and cellular impairment, resulting in a general decline of the physiological functions. The aim of this work was to evaluate age-related changes in circulating ROS levels and plasma protein carbonyls, in very young (2 months aged), young (8 months aged) and in middle age (15 months aged) F344 rats. In addition, the DNA oxidative marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) and the expression of the DNA repair enzymes APE1, OGG1 and UNG genes were also measured in the liver of these animals. We also determined whether systemic oxidative stress reflects oxidative injury at organ level. Our results demonstrate that the increase in circulating ROS and protein carbonyl content occurs as early as middle age. Moreover, increased 8-OHdG in the liver of 15-month-old rats was at least in part associated with a reduced DNA damage repairing capacity as suggested by the down-regulation of APE1 gene expression. In addition, we demonstrated for the first time, that plasma carbonyls and liver 8-OHdG are well correlated, suggesting that plasma protein carbonyls may be used as a surrogate marker of oxidative injury in target organs.

Pomegranate By-Products in Colorectal Cancer Chemoprevention: Effects in Apc-Mutated Pirc Rats and Mechanistic Studies In Vitro and Ex Vivo.

SCOPE: To investigate the effect of pomegranate mesocarp, a polyphenol-rich by-product of juice production, in colorectal cancer (CRC) chemoprevention. METHODS AND RESULTS: A mesocarp decoction (PMD) is administered for 6 weeks in the diet to Pirc rats, mutated in Apc, a key-gene in CRC. Mucin-depleted foci (MDFs), as CRC biomarkers, are reduced in PMD-fed rats compared to controls (MDF/colon: 34 ± 4 versus 47 ± 3, p = 0.02). There is an increase in apoptosis in MDFs from PMD-treated rats compared to controls (2.5 ± 0.2 versus 1.6 ± 0.2, p < 0.01). To elucidate the involved mechanisms, two colon-relevant metabolites of the polyphenolic and fiber PMD components, urolithin-A (u-A) and sodium butyrate (SB), are tested alone or in combination in vitro (colon cancer cells), and ex vivo in adenoma (AD) and normal mucosa (NM) from Pirc rats. u-A 25 µm plus SB 2.5 mm (USB) causes a significant reduction in COX-2 protein expression compared to untreated controls (about -70% in cancer cell cultures, AD, and NM), and a strong increase in C-CASP-3 expression in cells (about ten times), in AD and NM (+74 and +69%). CONCLUSION: These data indicate a chemopreventive activity of PMD due, at least in part, to pro-apoptotic and anti-inflammatory action of its metabolites that could be exploited in high-risk patients.

Astragali radix: could it be an adjuvant for oxaliplatin-induced neuropathy?

Neurotoxicity is a major side effect of platinum derivatives both during and after treatment. In the absence of effective pharmacological compounds, the opportunity to identify safe adjuvant treatments among medicinal plants seems appropriate. Astragali radix is an adaptogenic herbal product recently analyzed in platinum-treated cancer patients. With the aim of evaluating the anti-neuropathic profile of Astragali radix, a previously characterized aqueous (Aqu) and two hydroalcoholic (20%HA and 50%HA) extracts were tested in a rat model of oxaliplatin-induced neuropathy. Repeated administrations significantly reduced oxaliplatin-dependent hypersensitivity with 50%HA, the most effective, fully preventing mechanical and thermal hypersensitivity. Ex vivo, 50%HA reduced morphometric and molecular alterations induced by oxaliplatin in peripheral nerve and dorsal-root-ganglia. In the spinal cord and in brain areas, 50%HA significantly decreased activation of microglia and astrocytes. Furthermore, 50%HA prevented the nephro- and hepato-toxicity induced by the anticancer drug. The protective effect of 50%HA did not alter oxaliplatin-induced apoptosis in colon tumors of Pirc rats, an Apc-driven model of colon carcinogenesis. The hydroalcoholic extract (50%HA) of Astragali radix relieves pain and promotes the rescue mechanisms that protect nervous tissue from the damages triggering chronic pain. A safe profile strongly suggests the usefulness of this natural product in oxaliplatin-induced neuropathy.

Gene Expression Profile of Colon Mucosa after Cytotoxic Insult in wt and Apc-Mutated Pirc Rats: Possible Relation to Resistance to Apoptosis during Carcinogenesis.

Apc-mutated Pirc rats, spontaneously developing intestinal tumours, are resistant to 1,2-dimethylhydrazine- (DMH-) induced colon apoptosis. To understand this phenomenon, we analyzed the expression of genotoxic stress-related genes Mgmt, Gsta1, and Gstp1 in the colon of wt and Pirc rats in basal conditions and 24 h after DMH; plasmatic oxidant/antioxidant status was also evaluated. After DMH, Mgmt expression was increased in both genotypes but significantly only in wt rats; Gsta1 expression was significantly increased in both genotypes. Gstp1 expression did not vary after DMH but was lower in Pirc rats. Moreover, for each genotype, we studied by microarray technique whole gene expression profile after DMH. By unsupervised cluster analysis, 28 genes were differentially modulated between the two genotypes. Among them were interferon-induced genes Irf7, Oas1a, Oasl2, and Isg15 and the transcription factor Taf6l, overexpressed in DMH-treated wt rats and unchanged in Pirc rats. RT-PCR confirmed their overexpression in DMH-treated wt rats and showed a slighter variation in DMH-treated Pirc rats. Taken together, despite a blunted induction of Irf7, Oas1a, and Mgmt, defective apoptosis in Pirc rats 24 h after DMH is not mirrored by major differences in gene expression compared with wt rats.