Lifetime, low-dose ochratoxin A dietary study on renal carcinogenesis in male Fischer rats.

Carcinoma arising from male rat renal parenchyma is an aspect of the nephrotoxicity of ochratoxin A (OTA) and is a factor in considering application of animal data to human health risk assessment. We present experimental data to complement already published and to complete dose-response findings for dietary OTA. From 34 rats, only four unilateral renal carcinomas (12%) developed during a 2-year exposure to dietary OTA, contaminated to give the same weekly overall dosage as in the 50 µg kg(-1) gavage-dosing regimen of an NTP study (30%). Statistical analysis included adjustment for premature leukaemia deaths, resulting in the carcinoma incidence of 35% (10-81%), and showed no significant difference from NTP (incidence of 43% (23-49%)) due to the smaller number of animals. However, absence of microscopic neoplastic renal lesions in premature decedents argues for minimal effect of the 47% leukaemia on carcinoma expression in the present experiment. This would fit with previously published findings showing significantly less carcinoma expression from a regimen administering an OTA dose in feed than was achieved by a lower dose by gavage as in the NTP study. It is concluded that chronic gavage administration of OTA to male rats may optimise carcinoma incidence for toxicological purposes, but that the dietary mode gives data more applicable to assessing putative health risk for humans.

Kinetic impairment of haemopoietic stem cells in experimentally induced leukemia and aplastic anemia: an inverse correlation.

The production of blood cells from bone marrow (BM) hematopoietic stem cells (HSC) is regulated by a number of cytokines and growth factors that influence cell survival; differentiation, proliferation and apoptosis in health and supposedly, such mechanisms are deregulated in diseased conditions. As far as cellular kinetics is concerned HSCs are relatively quiescent in adults, have the ability to replicate symmetrically and asymmetrically and predictably exhibit multi-lineage hematopoietic reconstitution potential. HSC drive hematopoiesis and homeostasis by contracting and expanding the pool of hematopoietic cells in the bone marrow. In mouse they can be identified immunophenotypically as Sca1+ c-kit cells. In aplastic anemia a drastic decline in the marrow efficacy to produce mature blood cells leads to bone marrow failure. In contrast, in leukemia hyper stimulated marrow leads to deregulated differentiation of immature hematopoietic stem cells with increased self-proliferation potential. In our experimental set up, we induced aplastic anemia by injecting busulfan and cyclophosphamide and leukemia by N-N' ethylnitrosourea intraperitoneally in inbred swiss albino mice. Indeed, HSCs and haematopoietic progenitor cells (HPCs) are vulnerable target for such disease oriented dysregulation which bears close correlation with the bone marrow microenvironmental damage. The present study aims at evaluating the possible mechanism(s) of deregulation in the bone marrow physiology with special reference to HSC surface receptor expression, cellular granularity, cell cycle status and overall marrow architecture. The investigations made so far revealed an interesting correlation between disease initiation and specific cytokinetic involvement of HSC in the BM microenvironment with particular reference to leukemia and aplastic anemia.

Lack of promotion effects of 50 Hz magnetic fields on 7,12-dimethylbenz(a)anthracene-induced malignant lymphoma/lymphatic leukemia in mice.

New-born CD-1 mice were initiated with a single subcutaneous injection of 60 microg 7,12-dimethylbenz(a)anthracene (DMBA) within 24 h after birth. After weaning, the mice were randomly divided into five groups of 100, 50 males and 50 females each. One group served as a cage control. The other four groups of mice were exposed to either 0 (sham-exposed), 7, 70, or 350 microT(rms) circularly polarized 50 Hz magnetic fields (MFs) for 22 h/day, 7 days/week for 30 weeks. Animals were observed daily and the development of malignant lymphoma/lymphatic leukemia was examined histopathologically. The experiment was conducted twice. There was no observed sexual difference in the cumulative proportions of mice with malignant lymphoma/lymphatic leukemia and a 3-way analysis of deviance using the Cox regression model revealed no interactions between experiment, sex, or group. The cumulative proportions of mice with malignant lymphoma/lymphatic leukemia in the MF-exposed groups were not significantly higher than those in the sham-exposed group of each sex in individual experiments and in males and females combined in each experiment, and in all the animals from the two experiments combined. These data provide no evidence to support the hypothesis that power frequency MFs is a significant risk factor for hematopoietic neoplasia.

Leukemic transformation of hematopoietic cells in mice internally exposed to depleted uranium.

Depleted uranium (DU) is a dense heavy metal used in military applications. During military conflicts, US military personnel have been wounded by DU shrapnel. The health effects of embedded DU are unknown. Published data from our laboratory demonstrated that DU exposure in vitro can transform immortalized human osteoblast cells (HOS) to the tumorigenic phenotype. Results from our laboratory have also shown that DU is genotoxic and mutagenic in cultured human cells. Internalized DU could be a carcinogenic risk and concurrent alpha particle and heavy metal toxic effects complicate this potential risk. Anecdotal reports have suggested that DU can cause leukemia. To better assess this risk, we have developed an in vivo leukemogenesis model. This model involves using murine hematopoietic cells (FDC-P1) that are dependent on stimulation by granulocyte-macrophage colony stimulating factor (GM-CSF) or interleukin 3 (IL-3) and injected into mice to produce myeloid leukemia. Although immortalized, these cells are not tumorigenic on subcutaneous inoculation in mice. Intravenous injection of FDC-P1 cells into DU-implanted DBA/2 mice was followed by the development of leukemias in 76% of all mice implanted with DU pellets. In contrast, only 12% of control mice developed leukemia. Karyotypic analysis confirmed that the leukemias originated from FDC-P1 cells. The growth properties of leukemic cells from bone marrow, spleen, and lymph node were assessed and indicate that the FDC-P1 cells had become transformed in vivo. The kidney, spleen, bone marrow, muscle, and urine showed significant elevations in tissue uranium levels prior to induction of leukemia. These results demonstrated that a DU altered in vivo environment may be involved in the pathogenesis of DU induced leukemia in an animal model.

A new model of pre-B acute lymphoblastic leukemia chemically induced in rats.

OBJECTIVE: Although B acute lymphoblastic leukemia (B-ALL) is the most common leukemia among children, no chemically inducible model of this leukemia has yet been described in vivo. METHODS: Leukemia was chemically induced in male WKAH/Hkm rats by a nitrosourea derivative, N-butylnitrosourea (BNU), an alkylating agent, administered orally 5 days a week for 24 weeks. Development of leukemia was monitored by clinical observation, follow-up of blood parameters, and appearance of blast cells in peripheral blood samples. The phenotype of the leukemia was determined by cytological examination, cytochemical reactions, and by immunophenotyping of bone marrow cells using various markers. The feasibility of leukemia transplantation was investigated. Clonality and karyotype analyses were also performed. RESULTS: We observed the appearance of acute leukemia in 60% of the rats treated with BNU. Of these, 65% developed pre-B-ALL, which was serially transplantable to healthy WKAH/Hkm male rats. Karyotype analysis did not reveal clonal abnormalities. Clonality determined by immunoglobulin gene rearrangement sequencing disclosed that the pre-B-ALL were mostly oligoclonal. CONCLUSION: This new in vivo model of inducible pre-B-ALL might be useful for investigating the effects of co-initiating or promoting agents suspected to be involved in leukemia development, and for disclosing new molecular events leading to leukemogenic processes.

Evaluation of nonthreshold leukemogenic response to methyl nitrosourea in p53-deficient C3H/He mice.

The classic controversy of whether genotoxic chemicals induce cancers with or without a certain low-dose limit, i.e., the threshold, is revisited because of a number of current publications available addressing the plausibility of "practical" thresholds even for genotoxic carcinogens, the mechanism of which may be hypothesized to be due, in part, to a repair system composed of ordinarily available various defense mechanisms under the steady-state DNA damage. The question of whether an absolute nonthreshold or a relative nonthreshold, i.e., a "practical" threshold specifically in the low-dose level, is present may not be answered even with the use of a prohibitively large number of wild-type mice. Could the excessive incidence of tumorigenesis in p53-deficient mice contribute to our understanding of the threshold vs nonthreshold issue in genotoxic carcinogenesis? This is considered because an exaggeration of tumorigenesis in p53-deficient mice is hypothesized to reduce or eliminate the range of threshold due to the p53-deficiency-mediated reduction of DNA repair and apoptosis. The present study of chemical leukemogenesis in p53-deficient mice by transplantation assay was designed to answer this question. Briefly, 218 C3H/He mice were lethally irradiated and repopulated with bone marrow cells from wild-type, heterozygous p53-deficient, and homozygous p53-deficient C3H/He mice. This was followed by treatment with a single and graded dose of methyl nitrosourea at 6.6, 14.8, 33.3, 50.0, and 75.0 mg/kg body wt, with the vehicle-treated control groups treated with zero dose for each genotype. Whereas mice repopulated with p53-deficient bone marrow cells showed a marked reduction of the threshold for leukemogenicity, mice repopulated with wild-type bone marrow cells did not exhibit leukemia at a dose of 33.3 mg/kg body wt and showed a curve with a high probability for the linear regression model with a positive dose intercept, predicting a threshold by the likelihood ratio test. Thus, the failure of wild-type mice to show an increase in incidence of leukemogenesis at low doses of genotoxic carcinogens may be due not to a statistical rarity, but to various p53-related pharmacophysiological functions, possibly including DNA repair and apoptosis that may account for a threshold.

Flow cytometric analysis of DMBA-induced early in vivo ras expression.

According to recent publications 7,12-dimethylbenz[a]anthracene (DMBA) induces not only mammary cancer but also leukemia in Long-Evans (LE) rats. After treatment with DMBA, trisomy of the chromosome bearing N-ras and mutations in the codon 61 of different ras family genes are frequent. These alterations are already visible within 48 hours. Since there are very few data on ras genes' expression in the early stages of leukemogenesis, in our investigations LE rats were treated with DMBA and the expression of ras genes was measured within two days. DMBA was administered to outbred Long-Evans rats and the fluorescence intensity of the antibody recognizing the ras gene family was measured in femoral bone marrow cells 24 and 48 hours after the treatment. One of the bone marrow cell populations, separated by FSC and SSC, showed elevated ras gene expression at both 24 and 48 hours after the administration of the carcinogen. These results suggest that, besides the specific chromosomal aberrations and gene mutations, elevated ras gene expression could also be the marker of DMBA exposure.

Incidence of p53 and ras gene mutations in DMBA-induced rat leukemias.

Leukemia, a form of haematological malignancy, is a multi-stage disease and a wide range of diverse genes has been speculated to correlate with its initiation and development. Ras has been speculated to be an initiating gene for haematological malignancy, but more investigation will be needed to determine the genes associated with the progression of the disease. 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat leukemia provides a good tool for research into various stages of the disease. The entire coding regions of p53 and ras genes were examined for mutations in the present study. In this experiment, we used fluorescence-labeled polymerase chain reaction single-stranded conformation polymorphism analysis (PCR-SSCP) and direct sequencing to detect mutations of both genes on rat erythroleukemia. Fifteen out of 18 (83.3%) rat leukemias were found to have N-ras codon 61 mutation, consistent with previous results. The result of direct sequencing showed a single base substitution (CAA to CTA), resulting in an amino-acid change from Gln to Leu. No mutations were found in H-ras, K-ras or codon 12 of N-ras. The incidence of p53 gene mutation was 16.6% (3/18) in rat leukemia at late-stage. In the present study, mutation of the p53 gene was detected in three DMBA-induced leukemias as follows: a single-base substitution (CAT to CGT) at codon 177 (exon 5), resulting in an amino-acid change from Arg to Leu, a CGG to CTG/CGG changed at codon 211 (exon 6) resulting in an amino-acid change from His to Arg/His, and a GGG to TGG at codon 242 (exon 6) resulting in an amino-acid change from Gly to Trp, respectively. Thus, mutations of p53 gene do not seem to respond to the carcinogenesis of the DMBA-induced leukemia, in contrast to mutation of the N-ras oncogene, and may possibly be involved in the progress of multi-stage leukemogenesis.

Induction of stem cell cycling in mice increases their sensitivity to a chemical leukaemogen: implications for inherited genomic instability and the bystander effect.

Preconception paternal irradiation (PPI) modifies haemopoietic and stromal tissues of offspring and increases risk of generating lympho-haemopopietic malignancy if those offspring are then exposed to a leukaemogen. We hypothesised that this increased risk was related to inherited damage which had caused increased stem cell proliferation rates. To test for this link, in vivo, rapid stem cell proliferation was established by giving sub-lethal irradiation (3Gy gamma-rays) and allowing 3 days recovery. At this stage, 60% of haemopoietic spleen colony-forming units (CFU-S) were in DNA-synthesis, compared to <10% in unirradiated controls. Two groups of mice, unirradiated controls and irradiated animals, were then injected with 50mg/kg methyl nitrosourea (MNU) and observed daily for onset of lympho-haemopoietic malignancy. In a further control group of 60 mice, irradiated but not injected with MNU, only one leukaemia developed. In unirradiated controls, 20% of the mice developed malignancies between 3 and 8 months later: in the irradiated, MNU-treated groups, 95% developed malignancies between 2 and 7 months later. Thus, at least one powerful potentiating mechanism for induction of lympho-haemopoietc malignancy following inherited damage can be related to haemopoietic stem cell proliferation. Genomic instability is exposed by cell proliferation and has been implicated in this type of damage. However, a regulatory stromal microenvironment plays a part in inducing that proliferation. Thus, the microenvironment is the effective "bystander" which is thought to promote and amplify genomic instability, and thereby influence the induction of malignancy both in PPI offspring and in mice with induced stem cell proliferation.

Cytochrome P4501B1 mediates induction of bone marrow cytotoxicity and preleukemia cells in mice treated with 7,12-dimethylbenz[a]anthracene.

Humans are exposed to polycyclic aromatic hydrocarbons (PAHs) through many environmental pollutants, especially cigarette smoke. These chemicals cause a variety of tumors and immunotoxic effects, as a consequence of bioactivation by P-450 cytochromes to dihydrodiol epoxides. The recently identified cytochrome P4501B1 (CYP1B1) bioactivates PAHs but is also a physiological regulator, as evidenced by linkage of CYP1B1 deficiency to congenital human glaucoma. This investigation demonstrates that CYP1B1 null mice are almost completely protected from the acute bone marrow cytotoxic and preleukemic effects of the prototypic PAH 7,12-dimethylbenz[a]anthracene (DMBA). CYP1B1 null mice did not produce the appreciable amounts of bone marrow DMBA dihydrodiol epoxide DNA adducts present in wild-type mice, despite comparable hepatic inductions of the prominent PAH-metabolizing P-450 cytochrome, CYP1A1. Wild-type mice constitutively expressed low levels of bone marrow CYP1B1. These findings suggest that CYP1B1 is responsible for the formation of DMBA dihydrodiol epoxides in the bone marrow. Furthermore, this study substantiates the importance of DMBA dihydrodiol epoxide generation at the site of cancer initiation and suggests that tissue-specific constitutive CYP1B1 expression may contribute to cancer susceptibility in the human population.

Correlations among tumor types in mouse cancer bioassays: liver adenomas, liver carcinomas, leukemias and lymphomas.

In an examination of rodent bioassays Young and Gries [Young S.S., and Gries C.L. Exploration of the negative correlation between proliferative hepatocellular lesions and lymphoma in rats and mice--establishment and implications. Fundam. Appl. Toxicol. 1984: 4: 632-6401 and Haseman et al. [Haseman J.K., et al. Body weight-tumor incidence correlations in long-term rodent carcinogenicity studies. Toxicol. Pathol. 1997: 25: 256-263] noticed that there is a negative correlation (anticorrelation) between development of liver tumors and leukemia or lymphomas. If an animal has a lymphoma or leukemia it is less likely to develop liver tumors. These studies noted that this applies to several strains of animals. The anticorrelation appeared in control animals. In this paper we study this anticorrelation in the quarter of a million rodents exposed in the Carcinogenesis Bioassay Database System (CBDS) database of the National Toxicology program in both control and dosed animals. We failed to completely replicate Young and Gries or Haseman et al. However, when benign liver tumors (adenomas or nodules) and malignant liver tumors (carcinomas) are considered separately AND different leukemia and lymphoma types are considered, a strong anticorrelation appears. We identify survival and the time period (in years) during which the bioassay was completed as an important factor in interpreting correlations and anticorrelations. Differences between liver adenomas and carcinomas and lymphoma types contribute to a more general question of grouping of the individual tumor types. In our classification scheme (originally developed about 1986 by Dr. Bailar) an effort is made to distinguish benign and malignant neoplasms, while other investigators group all tumors at a specific site. For many analyses liver adenomas and carcinomas have been lumped together. This is because it is suspected that the diagnoses by pathologists may not distinguish, or ignore the (benign) adenoma when a (malignant) carcinoma is present. Possible biological differences in tumor mechanisms may require separate evaluation of these tumors with all the dangers of "pathologist bias" that this introduces.

Characterization of genetic instability in radiation- and benzene-induced murine acute leukemia.

This study, using the CBA/Ca mouse as a model, compares genetic lesions associated with radiation- and benzene-induced acute leukemias. Specific types of leukemia included in the analyses are radiation-induced acute myeloid leukemia (ML), and benzene-induced lymphoblastic leukemias, lymphomas, or mix-lineage leukemias. These leukemias have histopathological characteristics similar to those seen in human acute leukemias. G-band cytogenetic analysis showed that specific deletions involving regions D-E of one copy of mouse chromosome 2 [del(2)(D-E)] were frequently associated in both radiation- and benzene-induced acute leukemias. In addition, translocations of chr2(D-E) were also observed in some cases. These results suggest an important role of chr2 (D-E) deletions and translocations in the development of radiation- and benzene-induced murine acute leukemias. Fluorescence in situ hybridization with DNA probes specific for 2(D-E), constructed in our laboratory by means of chromosomal microdissection and PCR amplification, also demonstrate 2(D-E) deletions and/or translocations in these leukemic cells. Aneuploidy of chromosomes 3, 15, 16, and Y were also frequently detected in benzene-induced leukemic cells with or without lesions on chr2. These cytogenetic findings support the previous observations that metabolites of benzene lead to spindle-fiber disruption or abnormal cytokinesis in exposed animals. In summary, genetic instabilities observed in leukemic cells isolated from mice that had developed leukemia after exposure to radiation or benzene are syntenic with those frequently detected in patients with myelodysplastic syndrome, acute ML, and acute lymphoblastic leukemia. Thus, the CBA/Ca mouse has several characteristics that make it an excellent model for the study of radiation or benzene leukemogenesis in humans.

Review of mononuclear cell leukemia in F-344 rat bioassays and its significance to human cancer risk: A case study using alkyl phthalates.

Elevated incidences of mononuclear cell leukemia (MNCL) have been observed in a number of chronic bioassays in the F-344 rat. As this tumor type is unique to the rat and is only common in the F-344 strain, its significance for human cancer risk is unclear. For this reason, a survey of the published literature was undertaken to assess the occurrence and etiology of MNCL in F-344 rats and to evaluate its potential significance to humans using alkyl phthalate data as an example. It was found that MNCL occurs in untreated, aged F-344 rats at a high and variable rate, it is uncommon in most other rat strains, and its background incidence has increased significantly over time. This complicates retrospective data interpretation. MNCL has not been found in other mammalian species and no histologically comparable tumor is found in humans. In general, a statistically significant increase in frequency of a common tumor in the F-344 rat is an insufficient basis for determining that a chemical presents a carcinogenic hazard to humans, particularly when that tumor is not observed in other species. As one example, the alkyl phthalates constitute one group of substances which has been associated with increased MNCL frequency in the F-344 rat after high dietary doses. In evaluating the significance of this increase in MNCL, an extensive toxicological database for phthalates indicates that toxicological effects occur only at relatively high doses, and tumor development (including MNCL) occurs only after an apparent threshold is exceeded. Phthalates are not genotoxic as a class, further supporting the hypothesis of the existence of a threshold. When these considerations are collectively evaluated, it can be concluded that a finding of increased MNCL in F-344 rats exposed for a lifetime to a nongenotoxic chemical is not toxicologically relevant to humans, even when MNCL is observed at an increased incidence that is statistically significant. Thus, the increased incidence of MNCL observed in F-344 rats exposed to some alkyl phthalates is likely a strain-specific effect of little or no relevance for humans, and characterization of these chemicals as carcinogens based on increased MNCL in F-344 rats is not scientifically supported.

Effect of dietary curcumin and dibenzoylmethane on formation of 7,12-dimethylbenz[a]anthracene-induced mammary tumors and lymphomas/leukemias in Sencar mice.

Female Sencar mice (6 weeks old) were administered 1 mg of 7,12-dimethylbenz[a]anthracene (DMBA) by oral gavage once a week for 5 weeks. At 20 weeks after the first dose of DMBA, 68% of mice developed mammary tumors (the average 1.08 tumors per mouse) and 45% had lymphomas/leukemias. Feeding 1% dibenzoylmethane (DBM) in AIN 76A diet, starting at 2 weeks before the first dose of DMBA and continuing until the end of the experiment, inhibited both the multiplicity and incidence of DMBA-induced mammary tumor by 97%. The incidence of lymphomas/leukemias was completely inhibited by 1% DBM diet. In contrast, feeding 2% curcumin diet had little or no effect on the incidence of mammary tumors, and the incidence of lymphomas/leukemias was reduced by 53%.

A chimeric receptor/oncogene that can be regulated by a ligand in vitro and in vivo.

The BCR/ABL oncogene encodes an activated tyrosine kinase that causes human chronic myelogenous leukemia. The mechanism of transformation, however, is complex and not well understood. One of the important contributions of BCR to transformation is believed to be dimerization or oligomerization of ABL, thereby activating ABL tyrosine kinase activity. We reasoned that if ABL was dimerized through other mechanisms, activation of the tyrosine kinase activity should also result, and the activated kinase may also be transforming. Erythropoietin is known to activate its receptor by causing dimerization, and therefore a synthetic oncogene was created by linking the extracytoplasmic and transmembrane domains of the EPO receptor with c-ABL. This chimeric receptor was stably expressed in Ba/F3 cells and, in the absence of EPO, had no detectable biological effect on the cells. EPO, however, induced a rapid, dose-dependent activation of ABL tyrosine kinase activity and phosphorylation of several cellular proteins. The major target proteins have been identified, and are very similar to the known substrates of BCR/ABL, including Shc, CBL, CRKL, and several proteins in the cytoskeleton. EPO treatment also resulted in biological effects that were remarkably similar to those of BCR/ABL, including improved viability, altered integrin function, and a weak mitogenic signal. The biological effects were in part dose-dependent, in that low EPO concentrations enhanced viability but did not cause proliferation. At high EPO doses, kinase activation was maximal, and a mitogenic effect was also revealed. In nude mice, Ba/F3 cells expressing this chimeric receptor did not cause detectable disease without administration of pharmacologic doses of EPO. If EPO was given intraperitoneally 5 days a week, however, a dose-dependent lethal leukemia resulted. This ligand-regulatable oncogene mimics some of the biological effects of BCR/ABL, and analysis of ABL mutants in this system will be useful to dissect the signaling pathways that cause CML.