bluuebunny/crossref_metadata_2025 huggingface dataset:
High Frequency of Ras Mutations in Forestomach and Lung Tumors of B6C3F1 Mice Exposed to 1-Amino-2,4-dibromoanthraquinone for 2 Years
Authors: Hayashi Shim-Mo , Hong Hue-Hua L. , Toyoda Kazuhiro , Ton Thai-Vu T. , Devereux Theodora R. , Maronpot Robert R. , Huff James , Sills Robert C.
Published: January 2003

1-Amino-2,4-dibromoanthraquinone (ADBAQ) is an anthraquinone-derived vat dye, and a potent carcinogen in laboratory animals. In a 2-year study with dietary exposure to 10,000 or 20,000 ppm ADBAQ, increased incidence of forestomach and lung tumors were observed in B6C3F1 mice. The present study indentified genetic alterations in H- ras and K- ras proto-oncogenes in ADBAQ-induced tumors. Point mutations in ras proto-oncogenes were identified by restriction fragment length polymorphism, single-stranded conformational polymorphis m analysis and cycle sequencing of polymerase chain reaction-amplified DNA isolated from paraffin-embedded squamous cell papillomas and carcinomas in the forestomach, and alveolar/bronchiolar adenomas and carcinomas in the lung. A higher frequency of ras mutations was identified in ADBAQ-induced forestomach (23/32, 72%) and lung tumors (16/23, 70%) than in spontaneous forestomach (4/11, 36%) and lung tumors (26/86, 30%). H- ras codon 61 CTA mutations were detected in (4/8, 50%) ADBAQ-induced forestomach squamous cell papillomas and (10/24, 42%) squamous cell carcinomas, but not in the spontaneous forestomach tumors examined. H- ras codon 61 CGA mutation (6/24, 25%) was also detected in ADBAQ-induced forestomach squamous cell carcinomas. K- ras codon 61 A to T transversions and A to G transitions were prominent in ADBAQ-induced lung alveolar/bronchiolar adenomas and alveolar/bronchiolar carcinomas. The major finding of A to T transversions or A to G transitions in forestomach and lung tumors suggests that ADBAQ or its metabolites target adenine bases in the ras proto-oncogene s and that these mutations play a dominant role in multi-organ carcinogenesi s in the B6C3F1 mouse.
Di(2-ethylhexyl)phthalate Induces Hepatocellular Adenoma in Transgenic Mice Carrying a Human Prototype c-Ha-ras Gene in a 26-Week Carcinogenicity Study
Authors: Toyosawa Kaoru , Okimoto Kazuo , Kobayashi Izuru , Kijima Kazuyasu , Kikawa Emi , Kohchi Mami , Koujitani Takatoshi , Tanaka Kohji , Matsuoka Nobuo
Published: January 2003

To evaluate the transgenic mouse carrying a human prototype c-Ha- ras gene (rasH2 mouse) as a model for 26-week carcinogenicity tests, Di(2-ethylhexyl)phthalate (DEHP), a peroxisome proliferator, was administered to 15 rasH2 mice/sex/group at concentrations of 1,500, 3,000 or 6,000 ppm, and to 15 wild-type (non-Tg) mice/sex/group at a concentration of 6,000 ppm in their diets for 26 weeks. Survival rates and food consumption in the groups treated with DEHP and in the control group were similar. Body weight gain in rasH2 and non-Tg mice at 6,000 ppm in the terminal week decreased about 10% as compared to the control group. Common findings related to treatment with DEHP in rasH2 and non-Tg mice included hypertrophy with coarse granules and deposit of pigment in the liver, hydronephrosi s and tubular regeneration in the kidney, focal atrophy in the testis, and increased eosinophilic body in the nasal cavity. Hepatocellular adenoma was induced by treatment with DEHP, and was confined to male rasH2; mice the incidence being 7%(1/15), 13%(2/15), and 27%(4/15) in the 1,500-, 3,000-, and 6,000-ppm group, respectively. Point mutation was not detected in codon 12 and 61 of human c-Ha- ras transgene upon DNA analyses on frozen samples taken from these hepatocellular adenomas. From the results obtained in this 26-week carcinogenicity study, it is concluded that DEHP is a hepato-carcinogen for transgenic mouse carrying a human prototype c-Ha- ras gene.
Greater Expression of Transforming Growth Factor α and Proliferating Cell Nuclear Antigen Staining in Mouse Hepatoblastomas Than Hepatocellular Carcinomas Induced by a Diethylnitrosamine-Sodium Phenobarbital Regimen
Authors: Sakairi Tetsuya , Kobayashi Kiyoshi , Goto Kazuhiro , Okada Miyoko , Kusakabe Manami , Tsuchiya Takayuki , Sugimoto Jiro , Sano Fumiko , Mutai Mamoru
Published: January 2003

Transforming growth factor α (TGF- α) is a potent stimulator of normal hepatocyte proliferation, considered to have relationship to the liver regeneration or carcinogenesis. In this study, we investigated immunohistochemically the association between expression of TGF- α and cell proliferation activity in mouse hepatoblastoma s (HBs) and hepatocellular carcinomas (HCCs) induced in B6C3F1 mice by diethylnitrosamine and sodium phenobarbital. The TGF- α-positive rate in HBs (29.2%) was significantly higher than that in HCCs (12.7%). Likewise, the proliferating cell nuclear antigen-positive rate (22.2%) was higher than the HCC value (14.5%). On the individual data for both TGF- α and PCNA, most of the HBs showed higher positive rates than HCCs. In HBs, TGF- α was localized only in the nuclei, whereas some HCC cells stained positive both in their nuclei and cytoplasm (0.6%). These results suggest expression of TGF- α and its localization might be linked to cell proliferation and play a role in malignant progression of mouse HBs.
Reversibility of the Chronic Effects of Di(2-ethylhexyl)phthalate
Authors: David Raymond M. , Moore Michael R. , Finney Dean C. , Guest Derek
Published: January 2003

Fischer-344 rats treated with 12,500 ppm (728 and 879 mg/kg/d for male and females, respectively) and B6C3F1 mice treated with 6,000 ppm (1,227 and 1,408 mg/kg/d, respectively) di(2-ethylhexyl)phthalate (DEHP) in the diet for 78 weeks were allowed to recover for an additional 26 weeks on control diet. Blood was analyzed at weeks 78 and 104 from 10 animals per sex per group; animals were sacrificed at weeks 79 and 105 for histopathologic examination. The results are compared with data from animals continuously exposed to these dietary levels for 104 weeks (10, 11). Body weights and food consumption were measured monthly. BUN, albumin, and globulin that were significantly different for rats exposed to DEHP throughout 104 weeks, were comparable to controls for the recovery group. Reversibility of chronic effects on erythrocyte count, hemoglobin, and hematocrit values was apparent only for female rats. Chronic exposure demonstrated effects on liver, kidney, and testes weights. All organ weight effects except for testes for the Recovery group of rats, and all organ weight effects for mice, were reversible. Pigmentation of Kupffer cells and renal tubules present in chronically treated rats were not observed for the Recovery group. Lesions in the testes and pituitary gland were not reversible in rats. This may be a reflection of the senescence of the hypothalamic-gona d axis in rats. Cessation of exposure for mice resulted in amelioration of effects in the kidneys, liver, and testes. The extent of reversibility suggests that many chronic effects may be associated with a metabolic phenomenon such as peroxisome proliferation, which also reverted to control levels after 26 weeks of recovery.
A Retrospective Analysis of Background Lesions and Tissue Accountability for Male Accessory Sex Organs in Fischer-344 Rats
Authors: Suwa Takahiko , Nyska Abraham , Peckham John C. , Hailey James R. , Mahler Joel F. , Haseman Joseph K. , Maronpot Robert R.
Published: January 2003

Because the paired lobes (ventral, dorsal, lateral, and anterior) of the rat prostate have not been consistently sampled in many carcinogenicity and toxicity studies, comparison among different investigations has been compromised. The lack of specifi c site identifi cation for prostatic lesions further lessens the value of incidences reported. We present here the lobe-specific incidences and degree of severity of background prostatic, seminal vesicular, and ampullary glandular lesions in 1768 control Fischer-344 rats from 35 recent National Toxicology Program 2-year carcinogenicity and toxicity studies conducted in 4 laboratories. The dorsal and lateral lobes were combined and considered the dorsolateral lobe where inflammation, epithelial degeneration, mucinous cysts, and edema were observed. Infl ammation in the dorsolateral lobes was signifi cantly associated with pituitary gland adenoma whose prolactin was suggested to play an important role in pathogenesi s of prostatic inflammation. Epithelial degeneration, epithelial hyperplasia, infl ammation, edema, and adenoma were conspicuous in the ventral lobes. Infl ammation and edema occurred in the anterior lobes (coagulating glands). Inflammation, dilatation, epithelial hyperplasia, edema, and adenoma were observed in the seminal vesicles. Infl ammation was also present in the ampullary glands. We suggest an optimal embedment and trimming method in rat prostate and seminal vesicle to ensure adequate, consistent sampling.
Differential Effects of Transforming Growth Factor-β1, a Fibrogenic Factor, on Macrophage-Like Cells (HS-P) and Myofibroblastic Cells (MT-9) In Vitro
Authors: Yamate Jyoji , Maeda Masaru , Benn Sally J. , Laithwaite James E. , Allan Alison , Ide Mika , Kuwamura Mitsuru , Kotani Takao , Sakuma Sadashige , Lamarre Jonathan
Published: January 2003

Transforming growth factor- β1 (TGF- β1) produced by infiltrating macrophages plays a role in fibrotic disorders through the induction of myofibroblasts. To explore possible mechanisms by which TGF- β1 may act in this context, we investigated effects of TGF- β1 on macrophage-like (HS-P) and myofibroblastic (MT-9) cells, two novel cell lines developed by us. Immunocytochemicall y, the addition of TGF- β1 (0, 0.1, 0.5, and 1.0 ng/ml) dose-dependently suppressed the expressions of antigens recognized by macrophage/histiocyte-specific antibodies (ED1 and ED2) in HS-P cells, whereas the addition concomitantly increased the number of anti- α-smooth muscle actin antibody-positive myofibroblastic cells, suggesting a possible phenotypical modulation of macrophages into myofibroblasts in the fibrotic lesions. By contrast, MT-9 cells did not show such immunophenotypica l changes following TGF- β1 addition. DNA synthesis, measured by tritiated thymidine-incorporation , was inhibited in a dose-dependen tmanner in MT-9 cells by TGF- β1 addition (0, 0.1, 0.2, 0.5, 1.0, 5, and 10 ng/ml), but that in HS-P cells was unchanged. Northern blot analysis revealed that expressions of cell cycle-related early genes, c- jun and c- myc, were increased in HS-P cells after TGF- β1 (1 ng/ml) addition, with c- jun showing peak expression prior to c- myc. By contrast, the peak expressions of c- jun and c- myc were delayed in TGF- β1 (1 ng/ml)-added MT-9 cells, and their levels were less in MT-9 cells than in HS-P cells. Furthermore, TGF- β1 (1 and 10 ng/ml) induced DNA laddering in MT-9 cells, but did not in HS-P cells. Based on these findings, it was speculated that TGF- β1 could have induced G1 arrest in cell cycle and apoptosis in MT-9 cells. The present study showed that there were significant differences in the effects of TGF- β1 between macrophage-like HS-P cells and myofibroblastic MT-9 cells, presumably depending on divergent susceptibilities to TGF- β1 between both cell types. Because such cell types are key cells in the fibrogenesis, HS-P and MT-9 might be useful models for investigating the pathogenesis of fibrosis in vitro.
Assessment of Human Cancer Risk: Challenges for Alternative Approaches
Authors: Omenn Gilbert S.
Published: January 2003

The ILSI/HESI Workshopon Alternatives to Carcinogenicity Testing aims to develop and apply new methods for assessment of potential carcinogenic risk to humans from various chemicals. The Workshoprepresents a major cooperative scientific effort. The long-term goals should be to greatly enhance the effi ciency and reliability of such testing and to supplant, not just supplement, lifetime rodent bioassays. There are now well-established frameworks for risk assessment and risk management, putting risks into public health context and engaging stakeholders. The Lave-Omenn value-of-information model provides a useful way to assess the social costs and benefits of different strategies for testing large numbers of chemicals.
Criteria for the Evaluation of Studies in Transgenic Models
Authors: Popp James A.
Published: January 2003

The generation, evaluation, and presentation of data from the ILSI Alternatives to Carcinogenicity Testing (ACT) program was standardized to ensure that the results of studies performed in multiple laboratories could be reliably compared. To this end, standardized experimental protocols, tissue collection procedures, histopathology nomenclature, diagnoses, and terminology were employed by study participants. In the experimental phase, this approach provided important cross-model consistency. To ensure comparability in the data evaluation phase of the project, interpretive criteria were defi ned to allow the characterization of study outcome as positive, negative, or equivocal in regards to carcinogenic response. These criteria helped to provide consistency across models because separate Assay Working Groups were established to evaluate the results of each model. To organize and compile the data from the ILSI ACT program, a database has been developed and data entered in standardized format to facilitate cross- and intramodel comparisons. In summary, the early development of standardized test protocols, evaluation procedures, and interpretive criteria has resulted in a data set in which users can have a high level of assurance that results in the database refl ect consistently applied experimental and interpretive guidelines.
p53 +/- Hemizygous Knockout Mouse: Overview of Available Data
Authors: Storer Richard D. , French John E. , Haseman Joseph , Hajian Gerald , Legrand Edmund K. , Long Gerald G. , Mixson Lori A. , Ochoa Ricardo , Sagartz John E. , Soper Keith A.
Published: January 2003

The performance of the p53 +/- transgenic (knockout) mouse model was evaluated through review of the data from 31 short-term carcinogenicity studies with 21 compounds tested as part of the International Life Sciences Institute's (ILSI) Alternatives to Carcinogenicity Testing (ACT) project, together with data from other studies which used comparable protocols. As expected based on the hypothesis for the model, a significant number (12/16 or 75%) of the genotoxic human and/or rodent carcinogens tested were positive and the positive control, p-cresidine, gave reproducible responses across laboratories (18/19 studies positive in bladder). An immunosuppressive human carcinogen, cyclosporin A, was positive for lymphomas but produced a similar response in wild type mice. Two hormones that are human tumorigens, diethylstilbestrol and 17β-estradiol, gave positive and equivocal results, respectively, in the pituitary with p53-defi cient mice showing a greater incidence of proliferative lesions than wild type. None of the 22 nongenotoxic rodent carcinogens that have been tested produced a positive response but 2 compounds in this category, chloroform and diethylhexylphthalate, were judged equivocal based on effects in liver and kidney respectively. Four genotoxic noncarcinogens and 6 nongenotoxic, noncarcinogens were also negative. In total (excluding compounds with equivocal results), 42 of 48 compounds or 88% gave results that were concordant with expectations. The technical lessons learned from the ILSI ACT-sponsored testing in the p53+/- model are discussed.
The Tg.AC (v-Ha-ras) Transgenic Mouse: Nature of the Model
Authors: Tennant Raymond W. , Stasiewicz Stanley , Eastin William C. , Mennear John H. , Spalding Judson W.
Published: January 2003

The Tg.AC (v-Ha-ras) transgenic mouse model provides a reporter phenotype of skin papillomas in response to either genotoxic or nongenotoxic carcinogens. In common with the conventional bioassay, the Tg.AC model responds to known human carcinogens and does not respond to noncarcinogens. It also does not respond to most chemicals that are positive in conventional bioassays principally at sites of high spontaneous tumor incidence. The mechanism of response of the Tg.AC model is related to the structure and genomic position of the transgene and the induction of transgene expression through specific mediated interactions between the chemicals and target cells in the skin.
The rasH2 Transgenic Mouse: Nature of the Model and Mechanistic Studies on Tumorigenesis
Authors: Tamaoki Norikazu
Published: January 2003

The rasH2 mouse is a hemizygous transgenic mouse carrying the c-Ha-ras oncogene and that gene's promotor/enhancer within the genetic background of a BALB/cByJ C57BL/6J F1 mouse. Approximately 3 copies of the transgene are integrated in a tandem array into chromosome number 15. The transgene is transmitted stably without point mutation in hot spots and is expressed in all tissues over 20 backcross generations. The homozygous c-Ha-ras genotype is lethal. Hemizygotes are selected by polymerase chain reaction (PCR) analysis of tail tips after birth. Spontaneous tumors in hemizygous transgenic mice are rare until 6 months of age. The observed rasH2 tumor spectrum, including lung adenoma/adenocarcinoma, forestomach and skin papillomas, Harderian gland adenoma, liver proliferative lesions, splenic hemangioma/sarcoma, and lymphoma is consistent with the BALB/c and C57BL/6 background. In the rasH2 mouse, point mutations of the transgene induced by genotoxins are reported frequently but not in all tumors. Elevated levels of transgene expression were detected in all genotoxin-induced tumors in the rasH2. Increased transgene expression was independent of the mutation rate in transgenic and endogenous ras genes. These observations suggest that the overexpression of transgenic c-Ha-ras is responsible for accelerated tumor development.
DNA Repair—Deficient Xpa and Xpa/p53+/- Knock-Out Mice: Nature of the Models
Authors: Van Steeg Harry , De Vries Annemieke , Van Oostrom Conny TH. M. , Van Benthem Jan , Beems Rudolf B. , Van Kreijl Coen F.
Published: January 2003

Xeroderma pigmentosum (XP) is a rare autosomal recessive disease in which repair of ultraviolet (UV)-induced DNA damage is impaired or is totally absent due to mutations in genes controlling the DNA repair pathway known as nucleotide excision repair (NER). XP is characterized, in part, by extreme sensitivity of the skin to sunlight, and XP patients have a more than 1000-fold increased risk of developing cancer at sun-exposed areas of the skin. To study the role of NER in chemical-induced tumorigenesis in more detail, the authors developed Xpa-/- homozygous knockout mice with a complete defect in NER (designated as Xpa mice or XPA model). Xpa mice develop skin tumors at high frequency when exposed to UV light, and as such, they mimic the phenotype of human XP. Moreover, the Xpa mice also appear to be susceptible to genotoxic carcinogens given orally. Based on these phenotypic characteristics, the Xpa mice were considered to be an attractive candidate mouse model for use in identifying human carcinogens. In an attempt to further increase both the sensitivity and specifi city of the XPA model in carcinogenicity testing, the authors crossed Xpa mice with mice having a heterozygous defect in the tumor suppressor gene p53. Xpa/p53+/- double knockout mice develop tumors earlier and with higher incidences upon exposure to carcinogens as compared to their single knockout counterparts. Here the authors describe the development and features of the Xpa mouse and present some examples of the Xpa and Xpa/p53+/- mouse models' sensitivity towards genotoxic carcinogens. It appeared that the Xpa/p53 +/- double knockout mouse model is favorable over both the Xpa and p53+/- single knockout models in short-term carcinogenicity testing. In addition to the fact that the double knockout mice respond more robustly to carcinogens, they also appear to respond in a very discriminative way. All compounds identified thus far are true (human) carcinogens, and, therefore, the authors believe that the Xpa/p53+/- mouse model is an excellent candidate for a future replacement of the chronic mouse bioassay, at least for certain classes of chemicals.
Mechanisms of Hormonal Carcinogenesis in the p53+/- Hemizygous Knockout Mouse: Studies With Diethylstilbestrol
Authors: Carmichael Paul L. , Mills Jeremy J. , Campbell Mathew , Basu Maya , Caldwell John
Published: January 2003

The 2-year rodentbioassay has long had a central role in determining whether a compound is carcinogenic. It has recently been suggested that the use of 6-month studies in transgenic mice could reduce costs and animal numbers, without impairing the validity of cancer risk assessment. The p53 +/- hemizygous knockout mouse model is phenotypically stable and develops tumors during the 6-month study period only in response to chemical and physical stimuli, showing a high concordance with genotoxic rodent carcinogens. We treated p53+/- mice and wild-type parent strain (C57BL/6J) animals with diethylstilbestrol (DES), 500 μmol/kg i.p. for 4 days. Following sacrifice, DNA was extracted from various tissues and adducts measured by a modifi ed monophosphate version of the 32P-postlabelling assay. Major DES adducts were detected in the liver DNA of DES-treated wild-type mice at a level of 118.7 17.0 (mean SD relative adduct level [RAL]/10 10 nucleotides) compared with 207.7 36.4 in p53 +/-mice. No such adducts were detected in vehicle-treated animals. Total adduct levels, including endogenous I-compound adducts, in wild-type mice were 192.4 17.5 and 311.5 58.6 in p53 +/-animals. These data support the hypothesis that defi cient p53-dependent global genomic repair of DES adducts in p53+/-mice may result in the persistence of exogenous and endogenous DNA adducts that could contribute to earlier carcinogenicity in this model. We also prepared hepatic microsomes from male and female p53+/-and wild-type mice exposed to DES or vehicle. Western blot analysis demonstrated modestly higher basal levels of various cytochrome P450 (CYP) enzymes in the untreated p53 +/-mice compared to the wild-type mice. Furthermore, P450 levels were higher in female DES-treated p53 +/-mice compared to treated wild-type mice. For the p53 +/- knockout mice to be used with confi dence in drug safety studies, a further understanding of the metabolic capacity of these animals is needed.
Comparison of the Levels of Enzymes Involved in Drug Metabolism between Transgenic or Gene-knockout and the Parental Mice
Authors: Ariyoshi Noritaka , Imaoka Susumu , Nakayama Kazuo , Takahashi Yoshiki , Fujita Ken-Ichi , Funae Yoshihiko , Kamataki Tetsuya
Published: January 2003

Drug-metabolizing enzymes are involved in the metabolic activation or detoxification of carcinogens. To evaluate animals developed as models for alternative carcinogenicity testing, we investigated whether or not a gene manipulation including the transgene of ras and the knocking out of a tumor suppressor gene such as p53 orXPA could alter the expression of representative drug-metabolizing enzymes directly or indirectly. Expression of several isoforms of cytochrome P450 (CYP) in the liver of rasH2, p53 ( +/- ), Tg.AC, and XPA (-/-) mice with or without treatment of prototype inducer, phenobarbital or 3-methylcholanthrene, was analyzed by Western immunoblotting in comparison with their parental strains of mice. In addition, the activities of 3 major phase II enzymes, UDP-glucronosyltransferase, sulfotransferase, and glutathione S-transferase, were compared between the gene-manipulated and the corresponding parental strains of mice. Results demonstrate that XPA gene knockout appeared to increase constitutive expression of CYP2B and CYP3A isoforms. Overexpression of human c-Ha-ras gene or p53 gene knockout appeared to increase constitutive UGT activity toward 4-nitrophenol. The content or activities of almost all other enzymes examined in the present study do not appear to be affected by the gene manipulation.
A Perspective on Current and Future Uses of Alternative Models for Carcinogenicity Testing
Authors: Goodman Jay I.
Published: January 2003

This perspective is based upon the data presented at the International Life Sciences Institute (ILSI), Health and Environmental Sciences Institute Workshop on the Evaluation of Alternative Methods for Carcinogenicity Testing (ILSI Workshop). It is important to understand that all models discussed at the Workshop have limitations and that they are not designed to be employed as stand-alone assays. Although they may have other, appropriate applications, I do not recommend use of the SHE cell assay and the Tg.AC model for the regulatory purposes of a safety assessment. In my view, the neonatal mouse, p53 +/- , XPA -/- , XPA-/- and p53 +/-, and the rasH2 models can, as a component of an overall assessment, provide information on potential carcinogenicity of a chemical that is appropriate for consideration in a regulatory context. Generally, these models exhibit the ability to detect genotoxic compounds. In most cases these compounds would be detected in a standard battery of genotoxicity tests and, therefore, quite often the use of an alternative is not necessary. Actually, I believe that a bioassay in rats will suffi ce most of the time, that is, in my view, a routine bioassay in mice is not necessary. Specifi c circumstances where data obtained from one of the “recommended” alternative models might be helpful are discussed. With regard to lessons for the future, there is a particular need for models that are responsive to chemicals that exhibit a nongenotoxic mode of action. Additionally, new models will continue to be developed and their half-life will likely be substantially shorter than the time required for traditional validation. The development of enhanced paradigms for validation should be a priority so that improved safety assessment decisions can be made more quickly. However, while evaluating and validating such models, it is important to consider the fundamental issues, for example, rational dose selection, evaluation of mode of action in the context of dose-response relationships including the existence of thresholds and secondary mechanisms, and species-to-species extrapolation. The alternatives to carcinogenicity testing project was a very major undertaking. In addition to the valuable information provided, it serves to illustrate the value of cooperation between academia, government, and industry. Furthermore, the involvement of the International Life Sciences Institute as the overall organizing, facilitating umbrella was crucial for the success of the project.
Expectations for Transgenic Rodent Cancer Bioassay Models
Authors: Ashby John
Published: January 2003

The results of the present study have advanced dramatically the database on transgenic mouse abbreviated carcinogenicity bioassay models. As such, it will provide a secure foundationfor future evaluations of these assays and for their eventual validation as models for the prediction of possible human carcinogens. Based upon the results derived from the present study, it is suggested that 5 areas require discussion as a prelude to the further evaluation of existing models and the future evaluation of new models. First, there is the need to agree a standard list of calibration chemicals to be studied and to derive agreement on optimal bioassay group sizes, statistical methods, and exposure periods. Second, general agreement must be reached regarding the classes/types of known rodent carcinogens so that it is acceptable for the new models to fi nd negative, by implication, those rodent carcinogens considered not to pose a carcinogenic hazard to humans. Third, current understanding of mechanisms of carcinogenesis should be integrated into the evaluation of new bioassay models. Fourth, any changes made to the standard rodent carcinogenicity bioassay protocol will require compromises being made, and these should be commonly owned between interested parties in order to reduce the number of regional/agencyspecifi c carcinogenicity testing schemes. Fifth, a mechanism needs to be developed by which assays can be adopted or rejected for use in the routine bioassay of chemicals. In the absence of such initiatives the increasing number of new bioassay models will come to exist along side of the standard 2-species bioassay, and this may potentially lead to confusion regarding the true future role of these assays.
Panel Discussion on the Application of Alternative Models to Cancer Risk Assessment
Authors: Pettit Syril D.
Published: January 2003

The International Life Sciences Institute Alternative Carcinogenicity Testing (ILSI ACT) Workshopconcluded with a panel discussion that addressed the framework issue of the appropriate application of alternative models to human cancer risk assessment. This discussion encompassed both technical issues relating to the level of understanding of these models and their output as well as issues relating to the regulatory acceptance of these data. Although there were many different perspectives represented by the panelists, there was also significant consensus on many broad issues. This article focuses on several key areas of emphasis that were addressed by panelists and are considered critical issues for future discussions and evaluations.
Acute Dosage With Dexrazoxane, but not Doxorubicin, Is Associated With Increased Rates of Hepatic Protein Synthesis in vivo
Authors: Zima Tomas , Tesar Vladimir , Sherwood Roy , Sood Alka , Au Lai-Chong , Richardson Peter J. , Preedy Victor R.
Published: January 2003

An investigation was carried out into the effects of dexrazoxane and doxorubicin on hepatic protein synthesis in vivo. The protocol included 8 groups of rats and involved a pretreatment stage of 30 min followed by a treatment stage of either 2.5 or 24 h. Male Wistar rats (≅0.15-0.20 kg) were pretreated with either dexrazoxane (100 mg/kg; 5 ml/kg) or saline (0.15 mol/l NaCl; 5 ml/kg). At 30 min after the pretreatment, rats were again injected with either doxorubicin (5 mg/kg; 10 ml/kg) or saline (0.15 mol/l NaCl; 10 ml/kg) in the treatment phase. Rats were sacrifi ced at either 2.5 or 24 h after the last doxorubicin or saline injection. Rate of protein synthesis were measured 10 min prior to sacrifi cing rats, with a fl ooding dose of L-[4-3H]phenylalanine. Liver was analyzed for the protein synthetic capacity (Cs , mg RNA/g protein), the fractional rate of protein synthesis (ks, %/d), and the RNA activity (kRNA mg protein/d/mg RNA). Complementary analysis included plasma albumin, total protein and activities of alkaline phosphatase, and aspartate aminotransferase. In the 2.5-h study, doxorubici n alone had no effect on any of the above variables. Dexrazoxane alone increased Cs, ks and kRNA at 2.5 h. Combined dexrazoxane + doxorubicin increased hepatic CS and ks with concomitant reductions in total plasma protein. In the 24-h study, doxorubicin alone had no effect on any of the variables. Dexrazoxane alone had no effect on either CS, ks , or kRNA but raised plasma activities of alkaline phosphatase and aspartate aminotransferase. Combined dexrazoxane + doxorubicin increased Cs and ks and decreased total plasma protein and increased plasma aspartate aminotransferase activities at 24 h. In conclusion, there is no evidence that acutely doxorubicin per se has measurable effects on hepatic protein synthesis in vivo in an acute period. However, acutely dexrazoxane increases hepatic protein synthesis, which may represent its putative cytotoxic effects, as indicated by raised serum activities of liver enzymes. A combination of both dexrazoxane + doxorubicin appears to have a greater effect in increasing liver protein synthesis than dexrazoxane alone.
Evaluation of Diet and Dimethylarsinic Acid on the Urothelium of Syrian Golden Hamsters
Authors: Cano Martin , Arnold Lora L. , Cohen Samuel M.
Published: January 2003

Few studies have examined the carcinogenicity of chemicals toward the urinary bladder in hamsters, and the effect of diet on hamster urine and urothelium has not been reported. Our laboratory recently began investigating the effects of dimethylarsini c acid (DMA) on the hamster bladder, and we noticed subtle urothelial changes even in controls. The possible effect of various diets on hamster urothelium was evaluated by feeding different diets to 4-week-old Syrian Golden hamsters for 5 weeks. The diets examined were Tekland 8656, Purina 5002, Purina 5L79, and NIH-07. Light microscopic examination showed a slight increase in urothelial hyperplasi a in hamsters fed Purina 5L79. An increase in the incidence of urinary bladder necrosis, exfoliation, and mild hyperplasi a were noted by scanning electron microscopy (SEM) with all dietary preparation s except NIH-07. The constituents in the diets producing the urothelial alterations are not known at present, but NIH-07 diet was chosen for experiments to investigate the effects of DMA on the hamster bladder epithelium. Male and female 5-week-old Syrian Golden hamsters were fed 100 ppm DMA for 10 weeks. Examination of urinary parameters showed no treatment-related changes. Light microscopic examination and SEM revealed no changes of the urothelium of DMA-treated male or female hamsters.
Protein Kinase Activity Is Central to Rat Germ Cell Apoptosis Induced by Methoxyacetic Acid
Authors: Jindo Toshimasa , Wine Robert N. , Li Ling-Hong , Chapin Robert E.
Published: January 2003

Methoxyacetic acid (MAA) is a major metabolite of ethylene glycol monomethyl ether (EGME). Previous investigations of the testicular lesion induced by EGME have found that dividing meiotic cells are the most sensitive, although several stages of spermatocytes are also vulnerable. Preliminary data from this lab suggested the involvement of protein kinase activity in the development of this lesion, a hypothesis explored in the present studies. We used cultured seminiferous tubules (STs) from juvenile rats (25-day-old), exposed in vitro to MAA and several inhibitors of protein kinases. Nineteen h following a 5-h exposure to 5 mM MAA (the plasma level in vivo after a toxic dose of EGME), apoptotic spermatocytes were seen in early- and late-stage STs. Cell death was prevented by cotreatment with broad-spectrum inhibitors of protein kinases such as H-7, H-8, K-252a, W-7, and genistein. In corroboration, immunocytochemistry with antibodies to various kinases (PKC μ, ζ, and γ , AKAP220, CaMKII, MLCK, and Src) showed increased staining around dying spermatocytes following EGME treatment in vivo. 2D-PAGE, autoradiography, and nanospray mass spectrometry was used to separate and identify proteins whose phosphorylation status was most greatly changed following exposure to MAA. One protein was identified by sequence analysis as being glucose-regulated protein 94 (grp94). Western blotting and immunocytochemistry confi rmed this finding. The data we present implicate kinase activities in the pathogenesi s of this lesion and suggest the involvement of Sertoli cells.
Toxic Effects of Benzyl and Allyl Isothiocyanates and Benzyl-Isoform Specific Metabolites in the Urinary Bladder After a Single Intravesical Application to Rats
Authors: Masutomi Naoya , Toyoda Kazuhiro , Shibutani Makoto , Niho Naoko , Uneyama Chikako , Takahashi Noriyuki , Hirose Masao
Published: January 2003

Allyl isothiocyanate (AITC) is known to be weakly carcinogenic, whereas benzyl isothiocyanate (BITC) has been suggested to exert carcinogenicity toward the rat urinary bladder. To elucidate direct toxic effects of isothiocyanates (ITCs), BITC, AITC, or BITC-metabolites conjugated either with glutathione, cysteinylglycine, cysteine, or mercapturic acid were intravesically instilled into female F344 rats. Exposure to AITC and BITC at 2.8 mg/kg body weight, and the same mol quantity (37 μ mol/kg) of BITC-metabolites was for 2 h. Nineteen hours thereafter, the animals were intravenously administered 5-bromo-2'-deoxyuridine (BrdU) and killed 1 h later. BITC caused more profound toxic damage than AITC. Among the BITC-metabolites, cytotoxicity was evident with intermediate glutathione or cysteinylglycine conjugates, whereas the mercapturic acid, considered to be the major final urinary metabolite, exerted little effects. BrdU labeling was essentially dependent on the degree of cytotoxic potential of each compound. Considering the previous study results demonstrating the generation of free BITC from metabolites in urine, the present results support the idea that cytotoxic activity of orally administered ITCs is derived from free forms cleaved from conjugated metabolite(s) in urine.
Pathology of “Toxic Oils” and Selected Metals in the MRL/lpr Mouse
Authors: Koller Loren D. , Stang Bernadette V. , Posada de la Paz Manuel , Ruiz Mendez Maria V.
Published: January 2003

The Toxic Oil Syndrome epidemic that occurred in Spain in 1981 and affected nearly 20,000 people was caused by ingestion of oil mixtures that contained analine-denatured rapeseed oil. To date, an animal model in which to identify the actual etiologic agents(s) and to investigate the pathogenesi s of the disease has not been discovered. In this study, the MRL/lpr was used to assess the histopathological response of 3 “toxic oils” and 3 metals. The oils tested were a denatured rapeseed oil collected from a family who were affected by the Toxic Oil Syndrome epidemic in Spain (CO756) plus two synthesized oils (RSD and RSA). Female mice, 7 weeks of age, received an undiluted (neat) or a 1:10 diluted dose of each oil; mercury (50 ppm), cadmium (100 ppm), or lead (50 ppm). Half of each group was killed after 5 weeks of exposure and the remaining mice after 10 weeks of exposure. Body and organ weights (liver, kidney, thymus, and spleen) were recorded and selected organs were collected for histopathology. Ten weeks after treatment, body weights (BW) of the cadmium and lead groups were signifi cantly suppressed, and the body weight of the CO756-neat group was signifi cantly increased compared to their respective controls. Kidney/BW were decreased in the RSA-neat and RSA 1:10 groups after 10 weeks of exposure, and the kidney/BW in the mercury and cadmium groups were increased. Spontaneous development (12 weeks of age) and progression (17 weeks of age) of histopathological lesions are described for selected organs examined in the naïve mice as are changes that resulted from exposure to the “toxic oils” and metals. CO756-neat, mercury, and lead suppressed progression of the glomerulonephriti s that normally occurs in the MRL/lpr mouse. Also of interest were lesions that included mononuclear cuffi ng of hepatic bile ducts, progression of the granuloma s that formed in the renal glomeruli, vessels in the lymphoid organs that contained tightly packed lymphocytes, and the presence of plasma cells in the thymus. All 3 oils stimulated early development of the lymphoproliferative syndrome characteristic of the MRL/lpr mouse as demonstrated by an increase in the thymus/BW and spleen/BW ratios after 5 weeks of treatment. These data contribute to our knowledge of spontaneou s disease progression in the thymus, spleen, lymph nodes, and kidneys in the MRL/lpr mouse and the effects of 3 different “toxic oils” and metals on the development and progression of those lesions.
Review Article: Compendium of Chemical Carcinogens by Target Organ: Results of Chronic Bioassays in Rats, Mice, Hamsters, Dogs, and Monkeys
Authors: Swirsky Gold Lois , Manley Neela B. , Slone Thomas H. , Ward Jerrold M.
Published: January 2003

A compendium of carcinogenesi s bioassay results organized by target organ is presented for 738 chemicals that are carcinogeni c in chronic-exposure, long-term bioassays in at least 1 species. This compendium is based primarily on experiments in rats or mice; results in hamsters, monkeys, and dogs are also reported. The compendium can be used to identify chemicals that induce tumors at particular sites and to determine whether target sites are the same for chemicals positive in more than 1 species. The source of information is the Carcinogenic Potency Database (CPDB), which includes results of 6073 experiments on 1458 chemicals (positive or negative for carcinogenicity) that have been reported in Technical Reports of the National Cancer Institute/National Toxicology Program or in papers in the general published literature. The published CPDB includes detailed analyses of each test and citations. The CPDB is publicly available in several formats (http://potency.berkeley.edu). Chemical carcinogens are reported for 35 different target organs in rats or mice. Target organs in humans are also summarized for 82 agents that have been evaluated as human carcinogens at a particular target site by the International Agency for Research on Cancer (IARC). Comparisons are provided of target organs for mutagens versus nonmutagens and rats versus mice.
Pathology of Aging B6;129 Mice
Authors: Haines Diana C. , Chattopadhyay Sisir , Ward Jerrold M.
Published: January 2003

Fifty male and 49 female B6;129 mice (wild-type, + / + ) were maintained until 2 years of age to study their age-related pathology. By 104—105 weeks, 14/50 (28%) of the males and 30/49 (61%) of the females were still alive. The most common contributing cause of morbidity or mortality was lymphoma. Lymphoma was observed in 21/50 (42%) of the males and 33/49 (67%) of the females with the most common sites being mesenteric lymph nodes, gut associated lymphoid tissue (Peyer's patches), and spleen. The lymphoma most often appeared to arise in the mesenteric node. Immunohistochemistry revealed CD45R expression as well as infiltration by many CD3+ T cells. IgH gene rearrangements were found in typical mesenteric node lymphomas indicating B-cell origin. They bore similarities to the human T-cell rich, B-cell lymphomas. Other tumors included hepatocellular adenoma or carcinoma (male 12%, females 10%), lung alveolar Type II cell adenoma or carcinoma (male 32%, female 20%), thyroid follicular adenoma or carcinoma (male 2%, female 8%), ovarian tumors (17%), and endometrial tumors (6%). Nonneoplastic lesions included amyloidlike material in the nasal septum (male and female 100%), otitis media (male 84%, female 79%), epididymal epithelial karyomegaly (88%), melanosis (high incidences in various tissues including brain, parathyroid, and spleen), membranoproliferative glomerulonephritis (male 52%, female 71%), hyalinosis with extracellular crystals in several tissues (respiratory tract, gall bladder, stomach), islet cell hyperplasia (male 45%, female 29%) and esophageal dilation (male 10%, female 6%). The B6;129 mouse is a mouse with aging lesions similar to those in other mouse strains but with a characteristic common lymphoma.
Chronic Toxicity of Bromodichloromethane to the Japanese Medaka (Oryzias latipes)
Authors: Toussaint Margaret W. , Rosencrance Alan B. , Brennan Linda M. , Dennis William E. , Beaman Joseph R. , Wolfe Marilyn J. , Hoffmann Florence J. , Gardner Henry S.
Published: January 2003

Japanese medaka ( Oryzias latipes) were continually exposed in a flow-through diluter system for 9 months to measured bromodichloromethan e (BDCM) concentrations of 0.018, 0.143, or 1.424 mg/L. Parameters evaluated were hepatocarcinogenicity, hepatocellular proliferation, hematology, and intrahepatic BDCM concentration. BDCM was not hepatocarcinogeni c to medaka at the concentrations tested. Chronic toxicity was evidenced at 6 and 9 months by statistically significant ( α =0.05) levels of gallbladder lesions and bile duct abnormalities in medaka treated with 1.424 mg/L BDCM. Hepatocellular proliferation was assessed after 1, 4, and 20 days of BDCM exposure. Treatment-related increases or decreases in cellular proliferation were not observed at any time point. Hematocrit, leukocrit, cell viability, and cell counts of treated fish after 9 months of BDCM exposure were not significantly different from control fish. Intrahepatic concentrations were evaluated by gas chromatography after 9 months of BDCM exposure. Fish livers from all three BDCM treatments had detectable amounts of BDCM, with median intrahepatic concentrations of 1.02, 2.89, and 21.25 mg BDCM/kg fish liver in the low, middle, and high concentrations, respectively. Medaka chronic toxicity effects of statistically significant gallbladder and bile duct abnormalities occurred at 1.424 mg/L BDCM, well above median drinking water levels.
Mutant Models of Prolonged Life Span
Authors: Mahler Joel F.
Published: January 2003

Aging is an important biological process that affects all creatures. For humans, age-related diseases and the question of why we age and die also have tremendous social and philosophical impact. We can therefore expect that models to study mechanisms of the aging process will always attract much interest. Until recently, the mutant model approach to study molecular mechanisms of aging has been limited to lower animals such as yeast, worms, and flies. However, given the current power of genetic technology in mammals, we can expect that phenotypes of prolonged life span will increasingly be seen in mice and subject to evaluation by pathologists. A brief review of current models is presented.
Dynamics of nasopharyngeal colonization by potential respiratory pathogens
Authors: García-Rodríguez J. Á. , Fresnadillo Martínez M. J.
Published: January 2003

Studies have shown that colonization of the nasopharynx by potential respiratory pathogens Streptococcus pneumoniae , Haemophilus influenzae and Moraxella catarrhalis is established early in childhood, although rates vary greatly according to locality, sampling frequency, individual and social factors. Factors influencing colonization and elimination are not as yet fully understood, but adhesion to mucosal receptors and immune responses are implicated in addition to bacterial properties and colonization resistance dynamics. Colonization in children and adults has been intensively studied in various localities. Potential pathogens are more likely to colonize the nasopharynx of children prone to recurrent otitis media, where impaired local immunity and repeated exposure to respiratory pathogens are additional risk factors. Adults with chronic respiratory tract disease also have higher carriage rates. The factors contributing to increased risk of carriage of potential respiratory pathogens, as well as to clinical infection and antimicrobial resistance, are summarized in this review.
Buddhist economics and the environment
Authors: Daniels Peter L.
Published: January 2003

The reduction of the “metabolism” of the human economy has become one of the central themes of recent environmental and economic research and policy focused upon paths for achieving global sustainable development. Since the late 1980s, there has emerged a diverse array of “physical economy” approaches that utilise some form of material flow analysis (MFA) to quantify the pattern of flows of material and energy into, within, and out of the economic system. In principle, the reduction of the human socioeconomic metabolism, and appropriate changes in technology and consumption, are highly consistent with Buddhist economics. Indeed, MFA may be one of the most valuable devices for encouraging and implementing a global “green” technoeconomic paradigm that helps realize the type of benefits proffered under the vision of Buddhist economics. This paper describes the links between methodology or potential application of MFA and the central themes of the Buddhist economic path to the long‐term, harmonious co‐existence of humans within the natural environment.