Hprt Mutant Research Articles

A comparison of the genotoxicity of benzo[a]pyrene in four cell lines with differing metabolic capacity

Benzo[a]pyrene (B[a]P) is a known genotoxin and carcinogen, yet its genotoxic response at low level exposure has not been determined. This study was conducted to examine the interplay of dose and metabolic capacity on genotoxicity of B[a]P. Investigating and better understanding the biological significance of low level chemical exposures will help improve human health risk assessments. The genotoxic and mutagenic effects of B[a]P were investigated using human cell lines (AHH-1, MCL-5, TK6 and HepG2) with differential expression of the CYP450 enzymes CYP1A1, 1B1 and1A2 involved in B[a]P metabolism. MCL-5 and HepG2 cells showed detectable basal expression and activity of CYP1A1, 1B1 and 1A2 than AHH-1 which only show CYP1A1 basal expression and activity. TK6 cells showed negligible expression levels of all three CYP450 enzymes. In vitro micronucleus and HPRT assays were conducted to determine the effect of B[a]P on chromosome damage and point mutation induction. After 24h exposure, linear increases in micronucleus (MN) frequency were observed in all cell lines except TK6. After 4h exposure, only the metabolically competent cell lines MCL-5 and HepG2 showed MN induction (with a threshold concentration at 25.5μM from MCL-5 cells) indicating the importance of exposure time for genotoxicity. The HPRT assay also displayed linear increases in mutant frequency in MCL-5 cells, after 4h and 24h treatments. Mutation spectra analysis of MCL-5 and AHH-1 HPRT mutants revealed frequent B[a]P induced G to T transversion mutations (72% and 44% of induced mutations in MCL-5 and AHH-1 respectively). This study therefore demonstrates a key link between metabolic capability, B[a]P exposure time and genotoxicity.

Multi-walled carbon nanotubes (NM401) induce ROS-mediated HPRT mutations in Chinese hamster lung fibroblasts

Although there is an important set of data showing potential genotoxic effects of nanomaterials (NMs) at the DNA (comet assay) and chromosome (micronucleus test) levels, few studies have been conducted to analyze their potential mutagenic effects at gene level. We have determined the ability of multi-walled carbon nanotubes (MWCNT, NM401), to induce mutations in the HPRT gene in Chinese hamster lung (V79) fibroblasts. NM401, characterized in the EU NanoGenotox project, were further studied within the EU Framework Programme Seven (FP7) project NANoREG. From the proliferation assay data we selected a dose-range of 0.12 to 12µg/cm2 At these range we have been able to observe significant cellular uptake of MWCNT by using transmission electron microscopy (TEM), as well as a concentration-dependent induction of intracellular reactive oxygen species. In addition, a clear concentration-dependent increase in the induction of HPRT mutations was also observed. Data support a potential genotoxic/ carcinogenic risk associated with MWCNT exposure.

New investigations into the genotoxicity of cobalt compounds and their impact on overall assessment of genotoxic risk

The genotoxicity of cobalt metal and cobalt compounds has been widely studied. Several publications show induction of chromosomal aberrations, micronuclei or DNA damage in mammalian cells in vitro in the absence of S9. Mixed results were seen in gene mutation studies in bacteria and mammalian cells in vitro, and in chromosomal aberration or micronucleus assays in vivo. To resolve these inconsistencies, new studies were performed with soluble and poorly soluble cobalt compounds according to OECD-recommended protocols. Induction of chromosomal damage was confirmed in vitro, but data suggest this may be due to oxidative stress. No biologically significant mutagenic responses were obtained in bacteria, Tk+/− or Hprt mutation tests. Negative results were also obtained for chromosomal aberrations (in bone marrow and spermatogonia) and micronuclei at maximum tolerated doses in vivo. Poorly soluble cobalt compounds do not appear to be genotoxic. Soluble compounds do induce some DNA and chromosomal damage in vitro, probably due to reactive oxygen. The absence of chromosome damage in robust GLP studies in vivo suggests that effective protective processes are sufficient to prevent oxidative DNA damage in whole mammals. Overall, there is no evidence of genetic toxicity with relevance for humans of cobalt substances and cobalt metal.

Mutagenicity monitoring following battlefield exposures: Longitudinal study of HPRT mutations in gulf war i veterans exposed to depleted uranium

A total of 70 military Veterans have been monitored for HPRT T-cell mutations in five separate studies at 2-year intervals over an 8-year period. Systemic depleted uranium (DU) levels were measured at the time of each study by determining urinary uranium (uU) excretion. Each HPRT study included 30-40 Veterans, several with retained DU-containing shrapnel. Forty-nine Veterans were evaluated in multiple studies, including 14 who were in all five studies. This permitted a characterization of the HPRT mutation assay over time to assess the effects of age, smoking and non-selected cloning efficiencies, as well as the inter- and intra-individual variability across time points. Molecular analyses identified the HPRT mutation and T-cell receptor (TCR) gene rearrangement in 1,377 mutant isolates. An unexpected finding was that in vivo clones of HPRT mutant T-cells were present in some Veterans, and could persist over several years of the study. The calculated HPRT mutant frequencies (MFs) were repeatedly elevated in replicate studies in three outlier Veterans with elevated urinary uranium excretion levels. However, these three outlier Veterans also harbored large and persistent in vivo HPRT mutant T-cell clones, each of which was represented by a single founder mutation. Correction for in vivo clonality allowed calculation of HPRT T-cell mutation frequencies (MutFs). Despite earlier reports of DU associated increases in HPRT MFs in some Veterans, the results presented here demonstrate that HPRT mutations are not increased by systemic DU exposure. Additional battlefield exposures were also evaluated for associations with HPRT mutations and none were found.

Mutagenicity monitoring following battlefield exposures: Molecular analysis of HPRT mutations in Gulf War I veterans exposed to depleted uranium.

Molecular studies that involved cDNA and genomic DNA sequencing as well as multiplex PCR of the HPRT gene were performed to determine the molecular mutational spectrum for 1,377 HPRT mutant isolates obtained from 61 Veterans of the 1991 Gulf War, most of whom were exposed to depleted uranium (DU). Mutant colonies were isolated from one to four times from each Veteran (in 2003, 2005, 2007, and/or 2009). The relative frequencies of the various types of mutations (point mutations, deletions, insertions, etc.) were compared between high versus low DU exposed groups, (based on their urine U concentration levels), with HPRT mutant frequency (as determined in the companion paper) and with a database of historic controls. The mutational spectrum includes all classes of gene mutations with no significant differences observed in Veterans related to their DU exposures.

Fishing for radiation quality: chromosome aberrations and the role of radiation track structure.

The yield of chromosome aberrations is not only dependent on dose but also on radiation quality, with high linear energy transfer (LET) typically having a greater biological effectiveness per unit dose than those of low-LET radiation. Differences in radiation track structure and cell morphology can also lead to quantitative differences in the spectra of the resulting chromosomal rearrangements, especially at low doses associated with typical human exposures. The development of combinatorial fluorescent labelling techniques (such as mFISH and mBAND) has helped to reveal the complexity of rearrangements, showing increasing complexity of observed rearrangements with increasing LET but has a resolution limited to ∼10 MBp. High-LET particles have not only been shown to produce clustered sites of DNA damage but also produce multiple correlated breaks along its path resulting in DNA fragments smaller than the resolution of these techniques. Additionally, studies have shown that the vast majority of radiation-induced HPRT mutations were also not detectable using fluorescent in situ hybridisation (FISH) techniques, with correlation of breaks along the track being reflected in the complexity of mutations, with intra- and inter-chromosomal insertions, and inversions occurring at the sites of some of the deletions. Therefore, the analysis of visible chromosomal rearrangements observed using current FISH techniques is likely to represent just the tip of the iceberg, considerably underestimating the extent and complexity of radiation induced rearrangements.

Evaluation of vinyl laurate in a battery of in vitro and in vivo tests for genotoxicity

Vinyl laurate is a potential residual monomer in chewing gum base formulated with polyvinyl acetate vinyl laurate copolymer (PVAcVL). The genotoxic potential of vinyl laurate was examined in a battery of in vitro and in vivo genotoxicity tests. Vinyl laurate was not mutagenic in Ames tests. In addition, it was not mutagenic in the HPRT mutation assay in L5178Y cells. An in vitro mammalian chromosome aberration assay performed in CHO cells was equivocal. Vinyl laurate and/or its metabolites were not clastogenic in the mouse bone marrow micronucleus test. Kinetic data indicate that VL is metabolised to acetaldehyde and lauric acid. Both metabolites are well known and have been studied previously. Model calculations show, that any exposure to acetaldehyde from the consumption of PVAcVL containing chewing gum will remain far below levels of acetaldehyde exposure from food in which acetaldehyde occurs naturally. Direct exposure to VL will primarily be at the site of entry. The lack of toxicity in a 90-day repeated dose toxicity test, performed with VL doses up to approximately 3000 times higher than the maximal VL intake from the consumption of a typical piece of chewing gum, demonstrates a high safety margin.

Genotoxicity of 1-methylpyrene and 1-hydroxymethylpyrene in Chinese hamster V79-derived cells expressing both human CYP2E1 and SULT1A1.

1-Methylpyrene (1-MP) is a widespread pollutant that is carcinogenic in animals following metabolic activation. Previous studies have shown that benzylic hydroxylation of 1-MP, catalyzed by multiple CYP isoforms, gives rise to 1-hydroxymethylpyrene (1-HMP), which becomes bioreactive following further metabolism by various sulfotransferase (SULT) isoforms. However, the mutagenic and chromosome damaging effects of 1-MP and 1-HMP in mammalian cells have not been investigated. In this study a Chinese hamster V79-derived cell line expressing both human CYP2E1 and human SULT1A1 was used to investigate the ability of 1-MP and 1-HMP to induce cytotoxicity (using the CCK-8 assay), micronuclei and Hprt gene mutations. The role of each enzyme was investigated through co-exposure in the presence of an enzyme inhibitor. We found that at concentrations of 0.5-4 μM and 5-20 μM, under conditions where no reduction in cell viability/growth occurred, 1-HMP and 1-MP induced micronuclei in V79-hCYP2E1-hSULT1A1 cells in a concentration-dependent manner; however, both compounds were inactive in V79 cells. Similarly, they both caused an increase in Hprt mutant frequency in V79-hCYP2E1-hSULT1A1 cells in these concentration ranges, with 1-MP impairing cell viability/growth at 10 μM and above in the mutagenicity assay. The compounds were again both inactive in V79 cells. The effects of 1-HMP in V79-hCYP2E1-hSULT1A1 cells were blocked or reduced by addition of pentachlorophenol (PCP), a SULT1 inhibitor; the genotoxicity of 1-MP was significantly reduced by either 1-aminobenotrazole, a CYP2E1 inhibitor, or PCP. The results suggest that human CYP2E1 and SULT1A1 cooperate to activate 1-MP and cause genotoxicity in mammalian cells.

Nfkb1 is a haploinsufficient DNA damage-specific tumor suppressor.

NF-κB proteins play a central and subunit-specific role in the response to DNA damage. Previous work identified p50/NF-κB1 as being necessary for cytotoxicity in response to DNA alkylation damage. Given the importance of damage-induced cell death for maintenance of genomic stability, we examined whether Nfkb1 acts as a tumor suppressor in the setting of alkylation damage. Hprt mutation analysis demonstrates that Nfkb1−/− cells accumulate more alkylator-induced, but not ionizing radiation (IR)-induced, mutations than similarly treated wildtype cells. Subsequent in vivo tumor induction studies reveal that following alkylator treatment, but not IR, Nfkb1−/− mice develop more lymphomas than similarly treated Nfkb1+/+ animals. Heterozygous mice develop lymphomas at an intermediate rate and retain functional p50 in their tumors indicating that Nfkb1 acts in a haploinsufficient manner. Analysis of human cancers, including therapy-related myeloid neoplasms, demonstrates that NFKB1 mRNA expression is down regulated compared to control samples in multiple hematological malignancies. These data indicate that Nfkb1 is a haploinsufficient, pathway-specific tumor suppressor that prevents the development of hematologic malignancy in the setting of alkylation damage.

Once Upon a Mine: The Legacy of Uranium on the Navajo Nation

On a low, windswept rise at the southeastern edge of the Navajo Nation, Jackie Bell-Jefferson prepares to move her family from their home for a temporary stay that could last up to seven years. A mound of uranium-laden waste the size of several football fields, covered with a thin veneer of gravel, dominates the view from her front door. After many years of living next to the contamination and a litany of health problems she believes it caused, Bell-Jefferson and several other local families will have to vacate their homes for a third round of cleanup efforts by the U.S. Environmental Protection Agency (EPA). Decades of uranium mining have dotted the landscape across the Navajo Nation with piles of contaminated mine waste. The EPA has mapped 521 abandoned uranium mines on the reservation, ranging from small holes dug by a single prospector into the side of a mesa to large commercial mining operations.1 The Navajo people did not have a word for “radioactivity” when mining outfits looking for vanadium2 and uranium3 began moving onto their land in the 1940s, and they did not understand that radiation could be dangerous. They were not told that the men who worked in the mines were breathing carcinogenic radon gas and showering in radioactive water, nor that the women washing their husbands’ work clothes could spread radionuclides to the rest of the family’s laundry. Bell-Jefferson and her brother Peterson Bell played in and around the mines, splashing and swimming in pools of radioactive water that had been pumped out of the mines and then collected on their property. The contaminated water looked and tasted perfectly clean. Families used it for cooking, drinking, and cleaning. Hogans and corrals were built with mine wastes, as were roads. All that changed on 16 July 1979. Just about a mile and a half from Bell-Jefferson’s home, a dam broke at the United Nuclear Corporation mill, where workers processed ore from the nearby Northeast Church Rock uranium mine. The spill dumped 94 million gallons of mill process effluent and 1,100 tons of tailings—an acidic, radioactive sludge—into a large arroyo that emptied into the Puerco River.4 The Church Rock spill occurred less than four months after the partial meltdown of the Three Mile Island nuclear reactor, and it released three times as much radiation, making it the biggest nuclear spill in U.S. history, yet it received only a tiny fraction of the news coverage.5 Declared a Superfund site in 1983, the heaps of waste around the mill still cause radiation survey instruments to squeal from the invisible uranium atoms that remain active 30 years later.6 “This area used to be my playground,” Bell-Jefferson says. “Now it’s just a huge wound.” For the Bells and other Dine (the term by which many Navajo people refer to themselves), the Church Rock spill was a turning point. When corporate and government officials appeared in the spill’s aftermath and began inquiring into exposure to the slurry and potential health problems, the Navajo people finally learned the truth—far from being harmless, these uranium mines were poisoning people, and researchers say they will continue to do so for decades to come.

Is activation of the intra-S checkpoint in human fibroblasts an important factor in protection against UV-induced mutagenesis?

The ATR/CHK1-dependent intra-S checkpoint inhibits replicon initiation and replication fork progression in response to DNA damage caused by UV (UV) radiation. It has been proposed that this signaling cascade protects against UV-induced mutations by reducing the probability that damaged DNA will be replicated before it can be repaired. Normal human fibroblasts (NHF) were depleted of ATR or CHK1, or treated with the CHK1 kinase inhibitor TCS2312, and the UV-induced mutation frequency at the HPRT locus was measured. Despite clear evidence of S-phase checkpoint abrogation, neither ATR/CHK1 depletion nor CHK1 inhibition caused an increase in the UV-induced HPRT mutation frequency. These results question the premise that the UV-induced intra-S checkpoint plays a prominent role in protecting against UV-induced mutagenesis.

The Cellular and Molecular Carcinogenic Effects of Radon Exposure: A Review

Radon-222 is a naturally occurring radioactive gas that is responsible for approximately half of the human annual background radiation exposure globally. Chronic exposure to radon and its decay products is estimated to be the second leading cause of lung cancer behind smoking, and links to other forms of neoplasms have been postulated. Ionizing radiation emitted during the radioactive decay of radon and its progeny can induce a variety of cytogenetic effects that can be biologically damaging and result in an increased risk of carcinogenesis. Suggested effects produced as a result of alpha particle exposure from radon include mutations, chromosome aberrations, generation of reactive oxygen species, modification of the cell cycle, up or down regulation of cytokines and the increased production of proteins associated with cell-cycle regulation and carcinogenesis. A number of potential biomarkers of exposure, including translocations at codon 249 of TP53 in addition to HPRT mutations, have been suggested although, in conclusion, the evidence for such hotspots is insufficient. There is also substantial evidence of bystander effects, which may provide complications when calculating risk estimates as a result of exposure, particularly at low doses where cellular responses often appear to deviate from the linear, no-threshold hypothesis. At low doses, effects may also be dependent on cellular conditions as opposed to dose. The cellular and molecular carcinogenic effects of radon exposure have been observed to be both numerous and complex and the elevated chronic exposure of man may therefore pose a significant public health risk that may extend beyond the association with lung carcinogenesis.

Sensitivity of the Pig-a assay for detecting gene mutation in rats exposed acutely to strong clastogens

Clastogens are potential human carcinogens whose detection by genotoxicity assays is important for safety assessment. Although some endogenous genes are sensitive to the mutagenicity of clastogens, many genes that are used as reporters for in vivo mutation (e.g. transgenes) are not. In this study, we have compared responses in the erythrocyte Pig-a gene mutation assay with responses in a gene mutation assay that is relatively sensitive to clastogens, the lymphocyte Hprt assay, and in the reticulocyte micronucleus (MN) assay, which provides a direct measurement of clastogenicity. Male F344 rats were treated acutely with X-rays, cyclophosphamide (CP) and Cis-platin (Cis-Pt), and the frequency of micronucleated reticulocytes (MN RETs) in peripheral blood was measured 1 or 2 days later. The frequencies of CD59-deficient Pig-a mutant erythrocytes and 6-thioguanine-resistant Hprt mutant T-lymphocytes were measured at several times up to 16 weeks after the exposure. All three clastogens induced strong increases in the frequency of MN RETs, with X-rays and Cis-Pt producing near linear dose responses. The three agents also were positive in the two gene mutation assays although the assays detected them with different efficiencies. The Pig-a assay was more efficient in detecting the effect of Cis-Pt treatment, whereas the Hprt assay was more efficient for X-rays and CP. The results indicate that the erythrocyte Pig-a assay can detect the in vivo mutagenicity of clastogens although its sensitivity is variable in comparison with the lymphocyte Hprt assay.

Restoration of Mismatch Repair Functions in Human Cell Line Nalm-6, Which Has High Efficiency for Gene Targeting

Gene targeting is a powerful approach in reverse genetics. The approach has been hampered in most of human cell lines, however, by the poor targeting efficiency. Nalm-6, a human pre-B acute lymphoblastic leukemia cell line, exhibits exceptionally high gene targeting efficiency and is used in DNA repair and the related research fields. Nonetheless, usage of the cell line is still limited partly because it lacks expression of MSH2, a component of mismatch repair complex, which leads to increased genome instability. Here, we report successful restoration of MSH2 expression in Nalm-6 cells and demonstrate that the recovery does not affect the high targeting efficiency. We recovered the expression by introduction of cDNA sequences corresponding to exons 9 to 16 at downstream of exon 8 of the MSH2 gene. Endogenous exons 9 to 16 were deleted in the cell line. The MSH2 expression substantially reduced spontaneous HPRT mutation frequency. Moreover, gene targeting efficiency in the MSH2-expressing cells was similar to that in the MSH2-lacking cells. In fact, we generated heterozygously REV3L knockout and the catalytically dead mutants in the MSH2-proficient Nalm-6 cells with efficiency of 20–30%. The established cell line, Nalm-6-MSH+, is useful for reverse genetics in human cells.

Evaluating the weak in vivo micronucleus response of a genotoxic carcinogen, Aristolochic acids

Aristolochic acids (AAs) are carcinogenic plant toxins that are relatively strong gene mutagens, both in vitro and in vivo, but weak inducers of micronuclei in vivo. In order to clarify the reasons for these disparate responses, we evaluated the genotoxicity of AAs in F344 rats using several assays that respond to DNA damage in bone marrow. Groups of 7- to 8-week-old male rats (n=6) were gavaged with 0, 2.75, 5.5, and 11mg/kg AAs for 28 days or with 0, 11, 22, and 30mg/kg AAs for 3 days. Day 1 being the first day of treatment, Pig-a mutant frequencies (MFs) were assayed in peripheral blood erythrocytes up to Day 56 for the 28-day treatment or Day 42 for the 3-day treatment; micronuclei were assayed in peripheral blood reticulocytes on Day 4 (both treatment protocols) and on Day 29 of the 28-day treatment protocol; and at the final sampling times (Day 59 or Day 42), the animals were sacrificed and Hprt mutant lymphocytes were measured. In a separate study, the Comet assay was performed on liver, kidney, and bone marrow of animals gavaged with 0, 11, 22, and 30mg/kg AAs for 4 days and sacrificed 3h after the last treatment. While only weak increases in micronucleated reticulocyte frequency were observed in treated animals, Pig-a MFs increased in a dose- and time-dependent manner with both treatment schedules. Lymphocyte Hprt mutant frequencies also increased dose dependently in treated animals, and the Comet assay detected elevated levels of DNA damage in all the tissues evaluated. These findings indicate that the DNA damage produced by AAs in rat bone marrow is a weak inducer of micronuclei but a relatively strong inducer of gene mutation.

DNMTs are required for delayed genome instability caused by radiation

The ability of ionizing radiation to initiate genomic instability has been harnessed in the clinic where the localized delivery of controlled doses of radiation is used to induce cell death in tumor cells. Though very effective as a therapy, tumor relapse can occur in vivo and its appearance has been attributed to the radio-resistance of cells with stem cell-like features. The molecular mechanisms underlying these phenomena are unclear but there is evidence suggesting an inverse correlation between radiation-induced genomic instability and global hypomethylation. To further investigate the relationship between DNA hypomethylation, radiosensitivity and genomic stability in stem-like cells we have studied mouse embryonic stem cells containing differing levels of DNA methylation due to the presence or absence of DNA methyltransferases. Unexpectedly, we found that global levels of methylation do not determine radiosensitivity. In particular, radiation-induced delayed genomic instability was observed at the Hprt gene locus only in wild-type cells. Furthermore, absence of Dnmt1 resulted in a 10-fold increase in de novo Hprt mutation rate, which was unaltered by radiation. Our data indicate that functional DNMTs are required for radiation-induced genomic instability, and that individual DNMTs play distinct roles in genome stability. We propose that DNMTS may contribute to the acquirement of radio-resistance in stem-like cells.

Deregulated expression of DNA polymerase β is involved in the progression of genomic instability

Deregulated expression of DNA polymerase beta (pol β) has been implicated in genomic instability that leads to tumorigenesis, yet the mechanisms underlying the pol β-mediated genetic instability remain elusive. In this study, we investigated the roles of deregulated expression of pol β in spontaneous and xenobiotic-induced genetic instability using mouse embryonic fibroblasts (MEFs) that express distinct pol β levels (wild-type, null, and overexpression) as a model system. Three genetic instability endpoints, DNA strand breaks, chromosome breakage, and gene mutation, were examined under various expression levels of pol β by comet assay, micronuclei test, and hprt mutation assay. Our results demonstrate that neither pol β deficiency nor pol β overexpression is sufficient for accumulation of spontaneous DNA damage that promotes a hyperproliferation phenotype. However, pol β null cells exhibit increased sensitivity to exogenous DNA damaging agents with increased genomic instability compared with pol β wild-type and overexpression cells. This finding suggests that a pol β deficiency may underlie genomic instability induced by exogenous DNA damaging agents. Interestingly, pol β overexpression cells exhibit less chromosomal or DNA damage, but display a higher hprt mutation frequency upon methyl methanesulfonate exposure compared with the other two cell types. Our results therefore indicate that an excessive amount of pol β may promote genomic instability, presumably through an error-prone repair response, although it enhances overall BER capacity for induced DNA damage.

Combination effects of chronic cadmium exposure and gamma-irradiation on the genotoxicity and cytotoxicity of peripheral blood lymphocytes and bone marrow cells in rats

This work investigated the effects of chronic cadmium (Cd) exposure combined with γ-ray irradiation on the cytotoxicity and genotoxicity of peripheral blood cells and bone marrow cells in rats. Results showed that when the rats were exposed to low dose (LD) Cd of 0.1mg CdCl2/(kgd) for 8 and 12 weeks, the Cd concentration in blood reached to 135–140μg/L and no toxic effects on peripheral blood lymphocytes, white blood cells (WBC) and granulocyte–monocyte (GM) progenitor cells were observed except polychromatic erythrocytes (PCE) of bone marrow. Moreover, this chronic LD Cd exposure significantly decreased irradiation-induced micronucleus (MN) formation and hypoxanthine–guanine phosphoribosyl transferase (hprt) mutation in lymphocytes and PCE, while the combination of LD Cd exposure and irradiation induced the additive metallothionein (MT) protein expression in bone marrow cells. When the rats were exposed to a high dose (HD) Cd of 0.5mg CdCl2/(kgd) for 8 and 12 weeks, the blood Cd level approached to 458–613μg/L and an inflammatory response was induced, meanwhile, MN formation and hprt mutation were markedly increased, and the ratio of PCE/NCE (normochromatic erythrocyte) was significantly decreased. Furthermore, when the rats were exposed to HD Cd plus 2Gy irradiation, additive toxic effects on MN formation, hprt mutation, PCE damage and GM progenitor cell proliferation were observed, while this combination treatment resulted in an obvious reduction of MT protein compared to HD Cd group. In conclusion, chronic exposure to LD Cd induced the adaptive response to irradiation in the genotoxicity of peripheral blood lymphocytes and PCE of bone marrow by the up-regulation of Cd-induced MT protein, but the combination of HD Cd exposure and irradiation generated the additive effects on the cytotoxicity and genotoxicity in peripheral blood lymphocytes and bone marrow cells.

Report on stage III Pig‐a mutation assays using N‐ethyl‐N‐nitrosourea – comparison with other in vivo genotoxicity endpoints

N-Ethyl-N-nitrosourea (ENU) was evaluated as part of the Stage III trial for the rat Pig-a gene mutation assay. Groups of six- to eight-week-old male Sprague Dawley (SD) or Fischer 344 (F344) rats were given 28 daily doses of the phosphate buffered saline vehicle, or 2.5, 5, or 10 mg/kg ENU, and evaluated for a variety of genotoxicity endpoints in peripheral blood, spleen, liver, and colon. Blood was sampled predose (Day-1) and at various time points up to Day 57. Pig-a mutant frequencies were determined in total red blood cells (RBCs) and reticulocytes (RETs) as RBC(CD592-) and RET(CD592-) frequencies. Consistent with the results from a reference laboratory, RBC(CD592-) and RET(CD592-) frequencies increased in a dose and time-dependent manner, producing significant increases at all doses by Day 15, with similar frequencies seen in both rat strains. ENU also induced small but significant increases in % micronucleated RETs on Days 4 and 29. No significant increases in micronuclei were seen in the liver or colon of the ENU-treated SD rats. Hprt and Pig-a lymphocyte mutation assays conducted on splenocytes from Day 56 F344 rats detected two- to fourfold stronger responses for Hprt than Pig-a mutations. Results from the in vivo Comet assay in SD rats at Day 29 showed generally weak increases in DNA damage in all tissues evaluated. The results with ENU indicate that the Pig-a RET and RBC assays are reproducible, transferable, and complement other genotoxicity endpoints that could potentially be integrated into 28-day repeat dose rat studies.

An in vitro pig-A mutation assay? Could it be any better or easier than MLA or HPRT mutation assays?

An in vitro pig-A mutation assay? Could it be any better or easier than MLA or HPRT mutation assays?