Mutation Research 588 (2005) 58–63
Further evaluation of the skin micronucleus test: Results obtained using 10 polycyclic aromatic hydrocarbons
Takashi Nishikawa ?, Tsuneaki Nakamura, Akira Fukushima, Yutaka Takagi
Human Safety Evaluation Center, Lion, 100 Tajima, Odawara, Kanagawa 256-0811, Japan
Abstract The standard in vivo micronucleus (MN) test detects clastogenicity in hematopoietic cells and is not suitable for detecting chemicals that target the skin. Previously, we have developed an in vivo rodent skin MN test that is simple to perform and can be applied to several laboratory animals, including the hairless mouse—a species whose use simplifies the procedure of skin testing. In this paper, we report new data that confirms the predictive ability of the test. Following the application of 10 polycyclic aromatic hydrocarbons (7,12-dimethylbenz[a]anthracene; 3-methylcholanthrene; benzo[a]pyrene; dibenz[a,h]anthracene; benz[a]anthracene; dibenz[a,c] anthracene; chrysene; benzo[e]pyrene; pyrene; anthracene) with various degrees of genotoxicity to the dorsal skin of hairless mice, we found that these compounds caused MN production that in general correlated with their reported carcinogenicity. We believe that this test will be useful in detecting skin clastogens that test negative when analyzed using the standard micronucleus test.
Mutation Research 588 (2005) 35–46
Co-mutagenic activity of arsenic and benzo[a]pyrene in mouse skin
Jared M. Fischer a, Susan B. Robbins b, Mustafa Al-Zoughool c, Sasi S. Kannamkumarath d, Saundra L. Stringer a, Jon Scott Larson a, Joseph A. Caruso d, Glenn Talaska c, Peter J. Stambrook b, James R. Stringer a,?
a University of Cincinnati, Department of Molecular Genetics, Biochemistry and Microbiology, 231 Albert Sabin Way, Cincinnati, OH 45267-0524, USA
b University of Cincinnati, Department of Cell Biology, 3125 Eden Ave., Cincinnati, OH 45267-0521, USA
c University of Cincinnati, Department of Environmental Health, 3223 Eden Ave., Cincinnati, OH 45267-0056, USA
d University of Cincinnati, Department of Chemistry, 137 McMicken, Cincinnati, OH 45267-0037, USA
Abstract Exposure to inorganic arsenic in drinking water is linked to skin, lung and bladder cancer in humans. The mechanism of arsenicinduced cancer is not clear, but exposure to arsenic and polycyclic arylhydrocarbons (PAH) is more carcinogenic than exposure to either type of carcinogen alone. Arsenic can also generate reactive oxygen species, suggesting that oxidation of DNA may play a role in carcinogenesis. Oxidization of guanosines in polyG tracts is known to cause frameshift mutations, and such events can be detected in situ using the G11 placental alkaline phosphatase (PLAP) transgenic mouse model, which reports frameshift mutations in a run of 11 G:C basepairs by generating cells containing heat-resistant alkaline phosphatase activity. PAH can also induce frameshift mutations. In the study described here, FVB/N mice carrying the G11 PLAP transgene were crossed to C57Bl/6 mice. Half of the hybrid mice were given drinking water with sodium arsenite (10 mg/L) for 10 weeks. Half of the arsenic treated mice were also exposed to benzo[a]pyrene (BaP) by skin painting (500 nmol/week) for 8 weeks. Another group of mice was exposed to BaP but not arsenic. The effect on frameshift mutation was assessed by staining sections of skin tissue to detect cells with PLAP activity. Arsenic alone had no significant effect. On average, mice given BaP alone had approximately three times more PLAP-positive (PLAP+) cells. By contrast, mice exposed to both arsenic and BaP exhibited 10-fold more PLAP+ cells in the skin, and these cells were often arranged in large clusters, suggesting derivation from stem cells. Whereas combined treatment produced more PLAP+ cells, stable BaP adduct levels and arsenic burdens were not higher in mice exposed to both agents compared to mice exposed to either one agent or the other.
Reproductive Toxicology 20 (2005) 441–452
The Integrated Project ReProTect: A novel approach in reproductive
toxicity hazard assessment
Lars Hareng a, Cristian Pellizzer a, Susanne Bremer b, Michael Schwarz c, Thomas Hartung a,?
a European Centre for the Validation of Alternative Methods, Joint Research Center, Via Fermi 1, TP 580, 21020 Ispra (VA), Italy
b European Chemicals Bureau, Joint Research Center, Ispra, Italy
c Department of Toxicology, Institute of Pharmacology and Toxicology, University of Tuebingen, Tuebingen, Germany
Abstract Validated alternative test methods are urgently needed for safety testing of drugs, chemicals and cosmetics. Whereas some animal tests for topical toxicity have been successfully replaced by alternative methods, systemic toxicity testing requires new test strategies in order to achieve an adequate safety level for the consumer. Substantial numbers of animals are required for the current in vivo assays for drugs, chemicals and cosmetics and a broad range of pioneering alternative methods were already developed. These prerequisites motivate the development of a tiered testing strategy based on alternative tests for reproductive toxicity hazard. In the Integrated Project ReProTect, a consortium set up by the European Centre for the Validation of Alternative Methods (ECVAM) takes the lead to manage the development of a testing strategy in the area of reproductive toxicity. The reproductive cycle can be broken down into well-defined sub-elements, namely male and female fertility, implantation and pre/postnatal development. In this project, in vitro, in silico and sensor technologies will be developed, leading to testing strategies, that shall be implemented and disseminated
Regulatory Toxicology and Pharmacology 43 (2005) 35–44
Immune assessments in developmental and juvenile toxicology: Practical considerations for the regulatory safety testing of pharmaceuticals
Paul C. Barrow ¤, Guillaume Ravel
MDS Pharma Services, Les Oncins, 69210 Saint-Germain sur l’Arbresle, France
Abstract The developing organism is considered to be more sensitive than the adult to immunotoxic agents. There is every reason, therefore, to include immune assessments in the regulatory testing for developmental toxicity of drugs that are intended to be used in young patients or pregnant woman. An eVective strategy would be to incorporate immune assessments in the existing recommendations on pre- and post-natal toxicity study in the rat from the International Conference on Harmonisation. Immune assessments could also be included in juvenile toxicity studies to screen for eVects resulting from post-natal exposure to the drug. Adequate testing methods are available to screen for developmental eVects that result in immune depression. Routine immune assessments may comprise histopathological examination of the lymphoid organs/tissues and immunophenotyping of lymphocyte subsets in the blood, spleen, or thymus. These tests should be performed in rodents at various ages and at various stages of pre- and post-weaning development. Immunoglobulin and cytokine measurements, assessment of the T-cell dependent antigen response to sheep red blood cells or keyhole limpet haemocyanin antigens, and host resistance studies may be performed as apical tests at maturity. More research is required to develop methods for the detection of drugs that may render the developing organism more susceptible to hypersensitivity or autoimmunity.
Toxicology and Applied Pharmacology 207 (2005) S200 – S208
Design issues in toxicogenomics using DNA microarray experiment
Kyoung-Mu Leea,b,1, Ju-Han Kimc,1, Daehee Kanga,*
aDepartment of Preventive Medicine, Seoul National University College of Medicine, Institute of Environmental Medicine, SNUMRC, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, South Korea
bDepartment of Environmental Health, Graduate School of Public Health, Seoul National University, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, South Korea
cSeoul National University Biomedical Informatics (SNUBI), 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, South Korea
Abstract The methods of toxicogenomics might be classified into omics study (e.g., genomics, proteomics, and metabolomics) and population study focusing on risk assessment and gene–environment interaction. In omics study, microarray is the most popular approach. Genes falling into several categories (e.g., xenobiotics metabolism, cell cycle control, DNA repair etc.) can be selected up to 20,000 according to a priori hypothesis. The appropriate type of samples and species should be selected in advance. Multiple doses and varied exposure durations are suggested to identify those genes clearly linked to toxic response. Microarray experiments can be affected by numerous nuisance variables including experimental designs, sample extraction, type of scanners, etc. The number of slides might be determined from the magnitude and variance of expression change, false-positive rate, and desired power. Instead, pooling samples is an alternative. Online databases on chemicals with known exposure-disease outcomes and genetic information can aid the interpretation of the normalized results. Gene function can be inferred from microarray data analyzed by bioinformatics methods such as cluster analysis. The population study often adopts hospital-based or nested case-control design. Biases in subject selection and exposure assessment should be minimized, and confounding bias should also be controlled for in stratified or multiple regression analysis. Optimal sample sizes are dependent on the statistical test for geneto- environment or gene-to-gene interaction. The design issues addressed in this mini-review are crucial in conducting toxicogenomics study. In addition, integrative approach of exposure assessment, epidemiology, and clinical trial is required.