Rodent Carcinogenicity Research Articles

Disposition and metabolism of 2,2′-dimorpholinodiethyl ether in sprague dawley rats and B6C3F1/N mice after oral, intravenous administration, and dermal application

The specialty amine catalyst 2,2′-dimorpholinodiethyl ether (DMDEE) is a high-production volume chemical used in the production of flexible foam, high-resilient molded foam, and in coatings and adhesives. The disposition and metabolism of [14C]DMDEE (20 or 200 mg/kg) were determined in male ane female rats and mice after oral and intravenous administration and dermal application. In male and female rats, following a single oral administration, [14C]DMDEE was well-absorbed and excreted rapidly and extensively via urine (75–93%) and some in feces (∼4–8%). The total radioactivity in tissues at 24 h and 72 h (males only) following oral administration was 8–10% and ∼4%, respectively, suggesting considerable tissue distribution. A moderate amount of the total tissue radioactivity in kidney and liver were unextractable suggesting covalent binding of [14C]DMDEE-derived products in tissue macromolecules. Absorption following a single dermal application in rats was significant (∼64%) with a similar disposition pattern to oral. The oral and dermal disposition of [14C]DMDEE in male and female mice was similar to rats. Urinary products of DMDEE identified were oxidative metabolism of the morpholine ring. Coadministration of DMDEE with nitrite in rats didn’t produce the rodent carcinogen, N-nitrosomorpholine.

A review on the 40th anniversary of the first regulation of drinking water disinfection by-products.

Water disinfection, primarily by chlorination, is one of the greatest achievements of public health. However, more than half a century after its introduction, studies in the 1970s reported that (a) chlorine interacted with organic matter in the water to form disinfection by-products (DBPs); (b) two DBPs, chloroform and bromoform, both trihalomethanes (THMs), were rodent carcinogens; (c) three brominated THMs were mutagenic; in six studies chlorinated drinking waters in the United States and Canada were mutagenic; and (d) in one epidemiological study there was an association between bladder cancer mortality and THM exposure. This led the U.S. Environmental Protection Agency to issue its first DBP regulation in 1979. Forty years later, >600 DBPs have been characterized, 20/22 have been shown to be rodent carcinogens, >100 have been shown to be genotoxic, and 1000s of water samples have been found to be mutagenic. Data support a hypothesis that long-term dermal/inhalation exposure to certain levels of the three brominated THMs, as well as oral exposure to the haloacetic acids, combined with a specific genotype may increase the risk for bladder cancer for a small but significant population group. Improved water-treatment methods and stricter regulations have likely reduced such risks over the years, and further reductions in potential risk are anticipated with the application of advanced water-treatment methods and wider application of drinking water regulations. This 40-year research effort is a remarkable example of sustained cooperation between academic and government scientists, along with public/private water companies, to find answers to a pressing public health question.

Computational study of novel natural inhibitors targeting aminopeptidase N(CD13)

Objectives: To screen and identify ideal leading compounds from a drug library (ZINC15 database) with potential inhibition of aminopeptidase N(CD13) to contribute to medication design and development.Results: Two novel natural compounds, ZINC000000895551 and ZINC000014820583, from the ZINC15 database were found to have a higher binding affinity and more favorable interaction energy binding with CD13 with less rodent carcinogenicity, Ames mutagenicity, and non-inhibition with cytochrome P-450 2D6. Molecular dynamics simulation analysis suggested that the 2 complexes, ZINC000000895551-CD13 and ZINC000014820583-CD13, have favorable potential energy, and exist stably in the natural circumstances.Conclusion: This study discovered that ZINC000000895551 and ZINC000014820583 were ideal leading compounds to be inhibitions targeting to CD13. These compounds were selected as safe drug candidates as CD13 target medication design and improvement.Materials and Method: Potential inhibitors of CD13 were identified using a series of computer-aided structural and chemical virtual screening techniques. Structure-based virtual screening was carried out to calculate LibDock scores, followed by analyzing their absorption, distribution, metabolism, and excretion and toxicity predictions. Molecule docking was employed to reveal binding affinity between the selected compounds and CD13. Molecular dynamics simulation was applied to evaluate stability of the ligand-CD13 complex under natural environment.

Interleukin-6 selectively induces drug metabolism to potentiate the genotoxicity of dietary carcinogens in mammary cells

Breast cancer is the most commonly diagnosed malignancy in females, the etiology being multifactorial and includes the role of lifestyle exposure to DNA-damaging chemicals such as dietary carcinogens benzo (a) pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazo [4, 5-b] pyridine (PhIP). Both compounds require cytochrome P450 (CYP)-mediated metabolic activation to DNA-damaging species, and both induce transcriptional responses through the nuclear receptors Aryl hydrocarbon receptor (AhR) and estrogen receptor α (ERα). BaP and PhIP are mammary carcinogens in rodents. Clinically, circulating IL-6 expression is linked with poor prognosis of cancer and 35% of the deaths in breast cancer are linked with inflammation. The objective of this work was to investigate the molecular toxicology and local activation of BaP and PhIP in the presence of IL-6. Our laboratory has previously reported that miR27b can regulate CYP1B1 expression in colorectal cells, here we have investigated if this mechanism is working in mammary cell models, MCF-7 and MDA-MB-231 cells. Treatment (24 h) of cells with BaP (10 nM-10 µM) and PhIP (100 nM-100 µM) significantly induced genetic damage (micronuclei formation) in a dose-dependent manner in both cell lines. This effect was potentiated in the presence of human IL-6 at concentrations reported to be expressed in clinical breast cancer. On its own, IL-6 treatment failed to induce micronuclei frequency above the control levels in these cells. Compared to BaP or PhIP treatment alone, IL-6 plus BaP or PhIP selectively induced CYP1B1 significantly in both cell lines. Additionally, miR27b expression was downregulated by IL-6 treatments and transfection with miR27b inhibitor confirmed that miR27b is a regulator of CYP1B1 in both cell lines. These data show that BaP- and PhIP-induced DNA damage in mammary cells is potentiated by the inflammatory cytokine IL-6 and that inflammation-induced CYP expression, specifically CYP1B1 via miR27b, is responsible for this effect.

Molecular Dynamics Study of One-Base Deletion Duplexes Containing the Major DNA Adduct Formed by Ochratoxin A: Effects of Sequence Context and Adduct Ionization State on Lesion Site Structure and Mutagenicity.

Ochratoxin A (OTA) is a ubiquitous food toxin associated with chronic nephropathy in humans and renal carcinogenicity in rodents. The mutational spectra of cells exposed to OTA reveal that one-base deletions comprise the largest percentage (73%) of the total mutations that occur upon OTA exposure. To contribute toward understanding the prevalence of OTA-induced one-base deletion mutations, the present work uses molecular dynamics (MD) simulations to analyze the conformational preferences of one-base deletion duplexes containing OT-G, the major OTA adduct (addition product) at the C8-site of guanine. Specifically, the influence of OT-G in four possible ionization states and three sequence contexts (G1, G2 and G3 in the NarI (5'-G1G2CG3CC-3'), a prokaryotic mutational hotspot sequence) on the structure of the adducted DNA is investigated. Our data reveal that the damaged helices are stable in two (B-type (B) and stacked (S)) conformations that are structurally similar to those adopted by common N-linked C8-guanine lesions. However, the adduct ionization state and sequence context affect the degree of helical distortion and the B/S conformational heterogeneity, which will impact the lesion repair and replication outcomes. This finding correlates with the experimentally reported tissue-specific mutagenicity of OTA exposure. Furthermore, regardless of the adduct conformation, ionization state, or sequence context, more stable lesion-site interactions and lack of disruption of the flanking base pairs in the one-base deletion duplexes compared to the corresponding two-base deletion helices rationalize the greater abundance of OTA induced one-base deletions. Overall, our work provides valuable structural insights that help explain the experimentally observed mutagenicity associated with OTA.

Computational Study of Novel Natural Inhibitors Targeting O6-Methylguanine-DNA Methyltransferase

To screen ideal lead compounds from a drug library (ZINC15 database) with potential inhibition effect against O6-methylguanine-DNA methyltransferase (MGMT) to contribute to medication design and refinement. A series of computer-aided virtual screening techniques were used to identify potential inhibitors of MGMT. Structure-based virtual screening by LibDock was carried out to calculate LibDock scores, followed by absorption, distribution, metabolism, and excretion and toxicity predictions. Molecule docking was employed to demonstrate binding affinity and mechanism between the selected ligands and MGMT protein. Molecular dynamics simulation was performed to evaluate stability of the ligand-MGMT complex under natural circumstances. Two novel natural compounds, ZINC000008220033 and ZINC000001529323, from the ZINC15 database were found to bind with MGMT with a higher binding affinity together with more favorable interaction energy. Also, they were predicted to have less rodent carcinogenicity, Ames mutagenicity, and developmental toxicity potential as well as noninhibition with cytochrome P-450 2D6. Molecular dynamics simulation analysis demonstrated thatthe 2 complexes ZINC000008220033-MGMT and ZINC000001529323-MGMT had more favorable potential energy compared with reference ligand O6-benzylguanine, and they could exist stably in the natural environment. This study elucidated that ZINC000008220033 and ZINC000001529323 were ideal lead compounds with potential inhibition targeting to MGMT protein. These compounds were selected as safe drug candidates and may contribute a solid basis for MGMT target medication design and improvement.

Stilbene compound trans-3,4,5,4´-tetramethoxystilbene, a potential anticancer drug, regulates constitutive androstane receptor (Car) target genes, but does not possess proliferative activity in mouse liver

The constitutive androstane receptor(CAR) activation is connected with mitogenic effects leading to liver hyperplasia and tumorigenesis in rodents. CAR activators, including phenobarbital, are considered rodent non-genotoxic carcinogens. Recently, trans-3,4,5,4´-tetramethoxystilbene(TMS), a potential anticancer drug (DMU-212), have been shown to alleviate N-nitrosodiethylamine/phenobarbital-induced liver carcinogenesis. We studied whether TMS inhibits mouse Car to protect from the PB-induced tumorigenesis. Unexpectedly, we identified TMS as a murine CAR agonist in reporter gene experiments, in mouse hepatocytes, and in C57BL/6 mice in vivo. TMS up-regulated Car target genes Cyp2b10, Cyp2c29 and Cyp2c55 mRNAs, but down-regulated expression of genes involved in gluconeogenesis and lipogenesis. TMS did not change or down-regulate genes involved in liver proliferation or apoptosis such as Mki67, Foxm1, Myc, Mcl1, Pcna, Bcl2, or Mdm2, which were up-regulated by another Car ligand TCPOBOP. TMS did not increase liver weight and had no significant effect on Ki67 and Pcna labeling indices in mouse liver in vivo. In murine hepatic AML12 cells, we confirmed a Car-independent proapoptotic effect of TMS. We conclude that TMS is a Car ligand with limited effects on hepatocyte proliferation, likely due to promoting apoptosis in mouse hepatic cells, while controlling Car target genes involved in xenobiotic and endobiotic metabolism.

Computational study on new natural compound agonists of stimulator of interferon genes (STING)

ObjectiveThis study aimed to screen lead compounds and medication candidates from drug library (ZINC database) which has potential agonist effect targeting STING protein.Methods and materialsA series of computer-aided virtual screening techniques were utilized to identify potential agonists of STING. Structure-based screening using Libdock was carried out followed by ADME (absorption, distribution, metabolism, excretion) and toxicity prediction. Molecular docking was performed to demonstrate the binding affinity and mechanism between ligands and STING dimers. Molecular dynamic simulation was utilized to evaluate the stability of ligand-receptor complex. Finally, animal experiment was conducted to validate the effectiveness of selected compounds.ResultsThree novel natural compounds 1,2,3 (ZINC000015149223, ZINC000011616633 and ZINC000001577210, respectively) from the ZINC15 database were found binding to STING with more favorable interaction energy. Also, they were predicted with less ames mutagenicity, rodent carcinogenicity, non-developmental toxic potential and tolerant with cytochrome P450 2D6 (CYP2D6). The ligand chemical structure analysis showed the three compounds were inborn axisymmetric, such chemical structures account for combining and activating process of STING protein dimers. The dynamic simulation analysis demonstrated that ZINC000015149223-, ZINC000011616633- and ZINC000001577210-STING dimer complex had more favorable potential energy compared with amidobenzimidazole (ABZI) and they can exist in natural environments stably. Animal experiments also demonstrated that these three compounds could suppress tumor growth.ConclusionThis study demonstrates that ZINC000015149223, ZINC000011616633 and ZINC000001577210 are potential agonists targeting STING protein. These compounds are safe drug candidates and have a great significance in STING agonists development.

In vitro combinatory cytotoxicity of hepatocarcinogenic asarone isomers and flavonoids

Acorus calamus is a swamp herb, which is widely spread in northern hemisphere. It is used in infusions and in bitters but also in food supplements and in traditional herbal medicine. However, the main A. calamus ingredients, propenylic 2,4,5-trimethoxyphenylpropene isomers, termed alpha- (trans) and beta- (cis) asarone, are known carcinogens in rodents. Genotoxic and mutagenic properties are proposed.The presented in vitro cytotoxicity study focused on time-dependent and combinatory exposure scenarios. All experiments performed in HepG2 cells show moderate (in middle micromolar range) cytotoxicity with a time-dependent increase in effectiveness. The combination of the two asarone isomers in short time experiments (1 h) did not show any effect, whereas asarone isomer interaction changes from synergistic to antagonistic with an extended duration of exposure up to 72 h. The antagonism occurred predominantly in the naturally occurring trans/cis-asarone ratio of approximately 1:10. Combinatory cytotoxicity of asarones and selected, dietary relevant flavonoids in constant ratios was mainly attributed to flavonoid toxicity.

Population-Based Analysis of DNA Damage and Epigenetic Effects of 1,3-Butadiene in the Mouse.

Metabolism of 1,3-butadiene, a known human and rodent carcinogen, results in formation of reactive epoxides, a key event in its carcinogenicity. Although mice exposed to 1,3-butadiene present DNA adducts in all tested tissues, carcinogenicity is limited to liver, lung, and lymphoid tissues. Previous studies demonstrated that strain- and tissue-specific epigenetic effects in response to 1,3-butadiene exposure may influence susceptibly to DNA damage and serve as a potential mechanism of tissue-specific carcinogenicity. This study aimed to investigate interindividual variability in the effects of 1,3-butadiene using a population-based mouse model. Male mice from 20 Collaborative Cross strains were exposed to 0 or 635 ppm 1,3-butadiene by inhalation (6 h/day, 5 days/week) for 2 weeks. We evaluated DNA damage and epigenetic effects in target (lung and liver) and nontarget (kidney) tissues of 1,3-butadiene-induced carcinogenesis. DNA damage was assessed by measuring N-7-(2,3,4-trihydroxybut-1-yl)-guanine (THB-Gua) adducts. To investigate global histone modification alterations, we evaluated the trimethylation and acetylation of histones H3 and H4 across tissues. Changes in global cytosine DNA methylation were evaluated from the levels of methylation of LINE-1 and SINE B1 retrotransposons. We quantified the degree of variation across strains, deriving a chemical-specific human variability factor to address population variability in carcinogenic risk, which is largely ignored in current cancer risk assessment practice. Quantitative trait locus mapping identified four candidate genes related to chromatin remodeling whose variation was associated with interstrain susceptibility. Overall, this study uses 1,3-butadiene to demonstrate how the Collaborative Cross mouse population can be used to identify the mechanisms for and quantify the degree of interindividual variability in tissue-specific effects that are relevant to chemically induced carcinogenesis.

Cancer risk estimation of glycidol based on rodent carcinogenicity studies, a multiplicative risk model and in vivo dosimetry

Here we evaluate a multiplicative (relative) risk model for improved cancer risk estimation of genotoxic compounds. According to this model, cancer risk is proportional to the background tumor incidence and to the internal dose of the genotoxic compound. Furthermore, the relative risk coefficient per internal dose is considered to be approximately the same across tumor sites, sex, and species.In the present study, we demonstrate that the relative risk model is valid for cancer risk estimation of glycidol, a common food contaminant. Published tumor data from glycidol carcinogenicity studies in mice and rats were evaluated in combination with internal dose estimates from hemoglobin adduct measurements in blood from mice and rats treated with glycidol in short-term studies. A good agreement between predicted and observed tumor incidence in responding sites was demonstrated in the animals, supporting a relative risk coefficient that is independent of tumor site, sex, and species. There was no significant difference between the risk coefficients for mice (5.1% per mMh) and rats (5.4% per mMh) when considering internal doses of glycidol. Altogether, this mechanism-based risk model gives a reliable risk coefficient, which then was extrapolated to humans considering internal dose, and background cancer incidence.

Chances for reducing rodent carcinogenicity testing of human pharmaceuticals

Chances for reducing rodent carcinogenicity testing of human pharmaceuticals

The Antitussive Benzonatate Is Not Tumorigenic in Rodent Carcinogenicity Studies.

Benzonatate is a peripheral oral antitussive that dampens the activity of cough stretch receptors. Rodent carcinogenicity studies were performed in Tg.rasH2 mice and Wistar Han rats. Mice were orally gavaged benzonatate at 10, 30, 75, and 100 mg/kg/day for males and 5, 15, and 50 mg/kg/day for females. Rats were gavaged at 10, 30, and 90 mg/kg/day for males and 5, 15, and 50 mg/kg/day for females. Higher doses in males were due to differences in maximum tolerated doses in dose-ranging studies. In both species, benzonatate was not detected in plasma because of rapid ester hydrolysis producing 4-(butylamino) benzoic acid (BBA) and methylated polyethylene glycol polymer. This metabolism was similar in human plasma; therefore, plasma BBA was used to show systemic exposure. Both species had no evidence of a benzonatate-related increase in any neoplasm. A slight increase in nasal cavity exudative inflammation was present in benzonatate-dosed male mice. Retinal atrophy was observed in male rats at ≥30 mg/kg/day, but the incidence was within historical control data range and not related to benzonatate. In conclusion, benzonatate and its 2 major metabolites were not carcinogenic in rodent carcinogenicity studies at BBA exposures of ≥32 and 70 times a 200 mg human benzonatate dose, respectively.

Is oxidative stress involved in the sex-dependent response to ochratoxin A renal toxicity?

Ochratoxin A (OTA) is a mycotoxin considered the most powerful renal carcinogen in rodents and classified as a possible human carcinogen. Though its mechanism of action is still unknown, indirect DNA reactivity mediated by oxidative stress has been hypothesized to play an important role. Moreover, large sex-differences have been observed in carcinogenicity studies, male rats being more sensitive than females. Male and female F344 rats were administered (p.o.) with bicarbonate or 0.5 mg OTA/kg b.w. for 7 days; or with bicarbonate, 0.21 or 0.5 mg OTA/kg b.w. for 21 days. Total glutathione (tGSH) and oxidized glutathione (GSSG) levels, glutathione S-transferase (GST) and superoxide dismutase (SOD) activities were analysed in kidneys. The standard alkaline comet assay was used in combination with Formamidopyrimidine-DNA glycosylase (Fpg) to detect oxidized DNA bases in kidney. No biologically relevant sex-differences were observed in all the oxidative-stress related parameters analysed. Indeed, no relevant oxidative-stress related response was observed between treated animals and controls. In accordance with the similar OTA levels and histopathological changes between both sexes observed previously in the same animals, and with other oxidative-stress related parameters measured by others, results support that there are no differences between sexes in the oxidative stress response to OTA.

Metabolic Activation of the Cooked Meat Carcinogen 2-Amino-1-Methyl-6-Phenylimidazo[4,5-b]Pyridine in Human Prostate.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), an heterocyclic aromatic amine (HAA) formed in cooked meat, is a rodent and possible human prostate carcinogen. Recently, we identified DNA adducts of PhIP in the genome of prostate cancer patients, but adducts of 2-amino-3, 8-dimethylmidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-9 H-pyrido[2,3-b]indole (AαC), other prominent HAAs formed in cooked meats, were not detected. We have investigated the bioactivation of HAAs by Phase I and II enzymes in the human prostate (LNCaP) cell line using cytotoxicity and DNA adducts as endpoints. PhIP, MeIQx, and 2-amino-3-methylimidazo[4,5-f]quinoline, another HAA found in cooked meats, were poorly bioactivated and not toxic. The synthetic genotoxic N-hydroxylated-HAAs were also assayed in LNCaP cells with Phase II enzyme inhibitors. Notably, 2-hydroxy-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (HONH-PhIP), but not other HONH-HAAs, induced cytotoxicity. Moreover, PhIP-DNA adduct formation was 20-fold greater than adducts formed with other HONH-HAAs. Pretreatment of LNCaP cells with mefenamic acid, a specific inhibitor of sulfotransferase (SULT1A1), decreased PhIP-DNA adducts by 25%, whereas (Z)-5-(2'-hydroxybenzylidene)-2-thioxothiazolidin-4-one and pentachlorophenol, inhibitors of SULTs and N-acetyltransferases (NATs), decreased the PhIP-DNA adduct levels by 75%. NATs in cytosolic fractions of LNCaP cells and human prostate catalyzed DNA binding of HONH-PhIP by up to 100-fold greater levels than for SULT and kinase activities. Recombinant NAT2 is catalytically superior to recombinant NAT1 in the bioactivation of HONH-PhIP; however, the extremely low levels of NAT2 activity in prostate suggest that NAT1 may be the major isoform involved in PhIP-DNA damage. Thus, the high susceptibility of LNCaP cells recapitulates the DNA-damaging effect of HONH-PhIP in rodent and human prostate.

Genotoxicity assessment of the novel antitussive agent Benzonatate and its major metabolite

Benzonatate (TESSALON®) is a peripherally acting oral antitussive. It undergoes rapid ester hydrolysis producing 4-(butylamino) benzoic acid (BBA) and methylated polyethylene glycol (MPG) metabolites, which are eliminated in urine and feces. The nonclinical and clinical efficacy of Benzonatate has been demonstrated over the last 60 years, but its safety was not fully assessed. In this study, we tested the genotoxicity of Benzonatate and its major metabolite BBA in an in vitro bacterial reverse mutation and in vivo micronucleus assays. A chromosomal aberration assay was also performed on Benzonatate and BBA. In the reverse mutation assay, Benzonatate and BBA doses 1.5–5000 μg/plate ± S9 metabolic activation were used and the numbers of revertants/plate were compared to various controls. Chromosomal aberration assays with human peripheral blood lymphocytes used Benzonatate and BBA concentrations 25–2000 and 62.5–1930 μg/mL, respectively. A CByB6F1 mouse bone marrow micronucleus assay was performed as part of a 28-day oral toxicology study at up to 250 mg/kg/day. The frequencies of micronuclei in polychromatic erythrocytes in treated groups were compared with the control group. Neither Benzonatate nor BBA induced significant mutagenicity in any of the bacterial strains, with or without metabolic activation. They also did not produce any biologically relevant structural or numerical aberrations in human chromosomes. Benzonatate and its BBA and MPG metabolites rapidly produced from esterase activity did not produce any significant increase in the incidence of micronucleated polychromatic erythrocytes. In conclusion, Benzonatate and its major metabolite BBA were not mutagenic and did not cause numerical or structural chromosome alterations. While the MPG metabolite was not tested, studies on structural analogues indicated it was also unlikely to be genotoxic. This was supported by oral rodent carcinogenicity assays showing no increase in malignancies.

The transformics assay: first steps for the development of an integrated approach to investigate the malignant cell transformation in vitro.

The development of alternative methods to animal testing is a priority in the context of regulatory toxicology. Carcinogenesis is a field where the demand for alternative methods is particularly high. The standard rodent carcinogenicity bioassay requires a large use of animals, high costs, prolonged duration and shows several limitations, which can affect the comprehension of the human relevance of animal carcinogenesis. The cell transformation assay (CTA) has long been debated as a possible in vitro test to study carcinogenesis. This assay provides an easily detectable endpoint of oncotransformation, which can be used to anchor the exposure to the acquisition of the malignant phenotype. However, the current protocols do not provide information on either molecular key events supporting the carcinogenesis process, nor the mechanism of action of the test chemicals. In order to improve the use of this assay in the integrated testing strategy for carcinogenesis, we developed the transformics method, which combines the CTA and transcriptomics, to highlight the molecular steps leading to in vitro malignant transformation. We studied 3-methylcholanthrene (3-MCA), a genotoxic chemical able to induce in vitro cell transformation, at both transforming and subtransforming concentrations in BALB/c 3T3 cells and evaluated the gene modulation at critical steps of the experimental protocol. The results gave evidence for the potential key role of the immune system and the possible involvement of the aryl hydrocarbon receptor (AhR) pathway as the initial steps of the in vitro transformation process induced by 3-MCA, suggesting that the initiating events are related to non-genotoxic mechanisms.

Alternative Carcinogenicity Screening Assay Using Colon Cancer Stem Cells: A Quantitative PCR (qPCR)-Based Prediction System for Colon Carcinogenesis.

The carcinogenicity of chemicals in the environment is a major concern. Recently, numerous studies have attempted to develop methods for predicting carcinogenicity, including rodent and cell-based approaches. However, rodent carcinogenicity tests for evaluating the carcinogenic potential of a chemical to humans are time-consuming and costly. This study focused on the development of an alternative method for predicting carcinogenicity using quantitative PCR (qPCR) and colon cancer stem cells. A toxicogenomic method, mRNA profiling, is useful for predicting carcinogenicity. Using microarray analysis, we optimized 16 predictive gene sets from five carcinogens (azoxymethane, 3,2'-dimethyl-4-aminobiphenyl, N-ethyl-n-nitrosourea, metronidazole, 4-(n-methyl-n-nitrosamino)-1-(3-pyridyl)-1-butanone) used to treat colon cancer stem cell samples. The 16 genes were evaluated by qPCR using 23 positive and negative carcinogens in colon cancer stem cells. Among them, six genes could differentiate between positive and negative carcinogens with a p-value of < or =0.05. Our qPCR-based prediction system for colon carcinogenesis using colon cancer stem cells is cost- and time-efficient. Thus, this qPCR-based prediction system is an alternative to in vivo carcinogenicity screening assays.

Goodbye to the bioassay.

It is time to say goodbye to the standard two-year rodent bioassay. While a few, primarily genotoxic, compounds which are clearly associated with human cancer test positive in the bioassay, there is no science-based, sound foundation for presuming it provides either a valid broad (across different chemicals) capability for discerning potential human carcinogens or a valid starting point for making human risk assessment decisions. The two basic assumptions underlying the bioassay are: (1) rodent carcinogens are human carcinogens; and (2) results obtained at high doses are indicative of results that will occur at lower, environmentally relevant, doses. Both of these assumptions are not correct. Furthermore, a reevaluation of National Toxicology Program bioassay data has revealed that if the dose group size were increased from 50 to 200 rodents per group the number of bioassays deemed to be positive would increase from approximately 50% to very close to 100%. Thus, under the extreme conditions of the bioassay (e.g., high doses, lifetime exposure and, at times, a non-physiological route of administration) virtually all chemicals tested could be made into rodent carcinogens. In recent years there have been a number of proposals to move away from the standard bioassay. In particular, a recently formulated decision tree (Cohen, 2017), which places an emphasis on dose-response relationships and invites the use of MOA information, provides a sound basis for moving on from the bioassay and towards a rational approach to both identify chemicals which appear to have the potential to cause cancer in humans and take dose-response relationships into consideration in order to place the extent, if any, of the risk they might pose into proper perspective.

Case examples of an evaluation of the human relevance of the pyrethroids/pyrethrins-induced liver tumours in rodents based on the mode of action.

Rodent carcinogenicity studies are useful for screening for human carcinogens but they are not perfect. Some modes of action (MOAs) lead to cancers in both experimental rodents and humans, but others that lead to cancers in rodents do not do so in humans. Therefore, analysing the MOAs by which chemicals produce tumours in rodents and determining the relevance of such tumour data for human risk are critical. Recently, experimental data were obtained as case examples of an evaluation of the human relevance of pyrethroid (metofluthrin and momfluorothrin)- and pyrethrins-induced liver tumours in rats based on MOA. The MOA analysis, based on the International Programme on Chemical Safety (IPCS) framework, concluded that experimental data strongly support that the postulated MOA for metofluthrin-, momfluorothrin- and pyrethrins-produced rat hepatocellular tumours is mediated by constitutive androstane receptor (CAR) activation. Since metofluthrin and momfluorothrin are close structural analogues, reproducible outcomes for both chemicals provide confidence in the MOA findings. Furthermore, cultured human hepatocyte studies and humanized chimeric mouse liver studies demonstrated species difference between human hepatocytes (refractory to the mitogenic effects of these compounds) and rat hepatocytes (sensitive to their mitogenic effects). These data strongly support the hypothesis that the CAR-mediated MOA for liver tumorigenesis is of low carcinogenic risk for humans. In this research, in addition to cultured human hepatocyte studies, the usefulness of the humanized chimeric liver mouse models was clearly demonstrated. These data substantially influenced decisions in regulatory toxicology. In this review I comprehensively discuss the human relevance of the CAR-mediated MOA for rodent liver tumorigenesis based on published information, including our recent molecular research on CAR-mediated MOA.