Mutation induction by N-propyl-N-nitrosourea in eight MutaMouse organs.

As a part of the 2nd Collaborative Study for the Transgenic Mouse Mutation Assay, we studied the organ specificity and the temporal changes in mutant frequency (MF) of the lacZ gene following intraperitoneal injection of 250 mg/kg N-propyl-N-nitrosourea into male MutaMouse. We used a positive selection system and examined eight organs, i.e., bone marrow, liver, kidney, lung, spleen, brain, heart, and testis. The chemical caused a significant increase in MF in all organs except for brain, and the bone marrow was the most sensitive organ, exhibiting a MF on day 7 that was 10 times that of the control. The MF increased from day 7 to day 28 in liver, kidney, and testis, while it decreased in bone marrow. The relationship between the results of this study and the target organs of carcinogenesis, and the cause of the temporal changes in MF, are discussed.     

Target organ and time-course in the mutagenicity of five carcinogens in MutaMouse: a summary report of the second collaborative study of the transgenic mouse mutation assay by JEMS/MMS.

We studied five carcinogens for (a) organ-specific mutagenicity and expression time in the transgenic (TG) mouse mutation assay and (b) clastogenicity in the peripheral blood micronucleus assay in the same mice. Groups of mice were injected intraperitoneally (ip) with N-nitroso-di-n-propylamine (NDPA), propylnitrosourea (PNU), 7, 12-dimethylbenz[a]anthracene (DMBA), 4-nitroquinoline-1-oxide (4NQO), or procarbazine (PCZ); 4NQO was also administered orally. LacZ mutant frequencies (MF) of various organs, sampled 7, 14 and 28 days after treatment, were analyzed by galE positive selection. At least 5 organs were analyzed in each experiment. Bone marrow, liver, and testis were always analyzed, as were each chemical's target organs. All chemicals, except NDPA, induced micronuclei. All chemicals increased lacZ MF in all of their target organs for carcinogenesis and, to a lesser extent, in some non-target organs. That suggests that an organ that has a positive response to a chemical in the TG mouse mutation assay is likely to develop tumors on exposure to that chemical, but it does not always happen. The time-course of MF increases (7-28 days) differed among tissues. In general, time-dependent increase in MF occurred in organs with a low cell proliferation rate whereas no increase, or even a decrease, occurred in organs with a high proliferation rate. Our results demonstrated that the TG mouse mutation assay is effective for the detection of chemical mutagenesis in the target organs for carcinogenesis, and organ and time-course variations in chemical mutagenesis are important issues for the establishment of an optimal protocol for the assay.     

Multiple organ mutation in the lacZ transgenic mouse (Muta mouse) 6 months after oral treatment (5 days) with benzo[a]pyrene.

We have recently demonstrated that not all organs with high rates of mutation in the lacZ transgene develop tumors using the Muta Mouse. To better understand the role of in vivo mutation in carcinogenesis, we examined the mutant frequencies (MF) of the lacZ transgene in tumor-bearing and non tumor-bearing organs. MF, recovered after 2 weeks (the data taken from our previous study) and after 26 weeks following oral doses of 125 mg kg-1 day-1 benzo[a]pyrene (BP) for five days were compared. The organs examined included the target organs (forestomach, spleen, and lung) and non-target organs (colon, glandular stomach, and liver) for BP carcinogenesis. The data indicated that lacZ MF were markedly increased over spontaneous frequencies in the organs examined and that the organ which showed the highest MF was the colon, followed by the forestomach>spleen>glandular stomach, liver, and lung in that order. These findings indicate that the MF of the lacZ transgene in each organ, even 26 weeks after the start of the treatment does not fully correlate with the known target organs of BP. Furthermore, the lacZ MF in a non-papilloma region of a forestomach with a papilloma was equivalent to the two highest MF observed in the healthy colon (non-target organ) of mice at 26 weeks. These observations also indicate that the generation of tumors requires the induction of mutations as well as other factor(s) specific to the target organs. These results clearly suggest that highly mutated organs do not always progress to tumors in the transgenic mouse.     

In vivo genotoxicity evaluation of dimethylarsinic acid in MutaMouse.

Dimethylarsinic acid (DMA) induces DNA damage in the lung by formation of various peroxyl radical species. The present study was conducted to evaluate whether arsenite or its metabolite, DMA, could initiate carcinogenesis via mutagenic DNA lesions in vivo that can be attributed to oxidative damage. A transgenic mouse model, MutaMouse, was used in this study and mutations in the lacZ transgene and in the endogenous cII gene were assessed. When DMA was intraperitoneally injected into MutaMice at a dose of 10.6 mg/kg per day for 5 consecutive days, it caused only a weak increase in the mutant frequency (MF) of the lacZ gene in the lung, which was at most 1.3-fold higher than in the untreated control animals. DMA did not appreciably raise the MF in the bladder or bone marrow. Further analysis of the cII gene in the lung, the organ in which DMA induced the DNA damage, revealed only a marginal increase in the MF. Following DMA administration, no change in the cII mutation spectra was observed, except for a slight increase in the G:C to T:A transversion. Administration of arsenic trioxide (arsenite) at a dose of 7.6 mg/kg per day did not result in any increase in the MF of the lacZ gene in the lung, kidney, bone marrow, or bladder. Micronucleus formation was also evaluated in peripheral blood reticulocytes (RETs). The assay for micronuclei gave marginally positive results with arsenite, but not with DMA. These results suggest that the mutagenicity of DMA and arsenite might be too low to be detected in the MutaMouse.     

Genotoxicity of o-aminoazotoluene (AAT) determined by the Ames test, the in vitro chromosomal aberration test, and the transgenic mouse gene mutation assay

o-Aminoazotoluene (AAT) has been evaluated as a possible human carcinogen (Class 2B) by the International Agency for Research on Cancer (IARC). The Ames test found it to be mutagenic in the presence of a metabolic activation system, whereas it has little clastogenicity either in vitro or in vivo in the chromosomal aberration assay. AAT is also carcinogenic in the lung or liver of mice and rats given long-term administrations. Therefore, metabolites generated in the liver etc. may have gene mutation activity, and carcinogenesis would occur. We examined the mutagenicity of AAT in a gene mutation assay, using lacZ transgenic mice (MutaMice) and a positive selection method. AAT showed positive results for organs with metabolic functions, such as liver and colon and other organs. Positive results were also seen in an Ames test in the presence of metabolic activation and negative results seen in a chromosomal aberration test. Therefore, AAT had the potential to cause gene mutation in the presence of metabolic activation systems in vitro and the same reaction was confirmed in vivo with organs with metabolic function, such as liver and colon, but little clastogenicity in vitro or in vivo. Thus, metabolites with gene mutation activity may be responsible for the carcinogenicity of AAT. The transgenic mouse mutation assay proved to be useful for concurrent assessment of in vivo mutagenicity in multiple organs and to supplement the standard in vivo genotoxicity tests, such as the micronucleus assay which is limited to bone marrow as the only target organ.     

N-Nitrosodi-n-propylamine induces organ specific mutagenesis with specific expression times in lacZ transgenic mice.

The mutagenic and clastogenic effects of N-nitrosodi-n-propylamine (NDPA) in lacZ transgenic mice (MutaMouse) were investigated as a part of the second collaborative study of the transgenic mouse mutation assay by a subgroup of the Mammalian Mutagenesis Study Group, a suborganization of the Environmental Mutagen Society of Japan. Male MutaMouse mice were administered NDPA intraperitoneally at a dose of 250 mg/kg, which is half of the LD(50) of the compound. The clastogenicity of NDPA was examined by the peripheral blood micronucleus test just before and at 24, 48 and 72 h after the treatment. The mutant frequencies in the bone marrow, liver, lung, kidney and urinary bladder were examined by the positive selection method for lacZ kidney. These findings demonstrate that NDPA induces organ-specific mutagenesis with specific expression times, and that the mutagenicity of NDPA in lacZ transgenic mice is consistent with its carcinogenicity.     

Mutagenicity of 4-nitroquinoline 1-oxide in the MutaMouse.

As part of a collaborative study, the Mammalian Mutagenesis Study Group (MMS), a sub-organization of the Environmental Mutagen Society of Japan (JEMS) conducted mutagenicity tests in MutaMouse. Using a positive selection method, we studied the organ-specificity and time dependence of mutation induction by 4-nitroquinoline 1-oxide (4NQO). A single dose of 4NQO was administered intraperitoneally (7.5 or 15 mg/kg) or orally (200 mg/kg) to groups of male mice. On days 7, 14 and 28 after treatment, we isolated the liver, kidney, lung, spleen, bone marrow, testis and stomach in the intraperitoneal administration experiment and the liver, lung, bone marrow, testis and stomach in the oral administration experiment. In addition, we performed the peripheral blood micronucleus test to evaluate clastogenicity. In the mice treated intraperitoneally at 7.5 mg/kg, we found increased mutant frequency (MF) only in the lung, where the MF did not vary with expression time. In the mice treated at 15 mg/kg, we found increased MF in the liver, bone marrow and lung. In orally treated mice, the MF was high in the lung and liver and very high in the bone marrow and stomach while the increase in the testis was negligible. As the expression time was prolonged, the MF tended to increase in the liver, decrease in the bone marrow, and remain stable in the lung, testis and stomach. The incidence of micronucleus induction in peripheral blood cells was significantly increased (p<0.01) in the 4NQO groups when compared with the vehicle control group by intraperitoneal treatment. Thus, these assay systems appeared to be of use in detecting not only genetic mutation but also chromosomal aberration.     

Mutagenicity testing with transgenic mice. Part I: Comparison with the mouse bone marrow micronucleus test

As part of a larger literature study on transgenic animals in mutagenicity testing, test results from the transgenic mutagenicity assays (lacI model; commercially available as the Big Blue(R) mouse, and the lacZ model; commercially available as the Mutatrade markMouse), were compared with the results on the same substances in the more traditional mouse bone marrow micronucleus test. 39 substances were found which had been tested in the micronucleus assay and in the above transgenic mouse systems. Although, the transgenic animal mutation assay is not directly comparable with the micronucleus test, because different genetic endpoints are examined: chromosome aberration versus gene mutation, the results for the majority of substances were in agreement. Both test systems, the transgenic mouse assay and the mouse bone marrow micronucleus test, have advantages and they complement each other. However, the transgenic animal assay has some distinct advantages over the micronucleus test: it is not restricted to one target organ and detects systemic as well as local mutagenic effects.     

Nitrogen-substitution effect on in vivo mutagenicity of chrysene

We have previously reported the in vivo mutagenicity of aza-polycyclic aromatic hydrocarbons (azaPAHs), such as quinoline, benzo[f]quinoline, benzo[h]quinoline, 1,7-phenanthroline and 10-azabenzo[a]pyrene. The 1,10-diazachrysene (1,10-DAC) and 4,10-DAC, nitrogen-substituted analogs of chrysene, were shown to exhibit mutagenicity in Salmonella typhimurium TA100 in the presence of rat liver S9 and human liver microsomes in our previous report, although DACs could not be converted to a bay-region diol epoxide, the ultimate active form of chrysene, because of their nitrogen atoms. In the present study, we tested in vivo mutagenicity of DACs compared with chrysene using the lacZ transgenic mouse (Mutatrade markMouse) to evaluate the effect of the nitrogen substitution. DACs- and chrysene-induced mutation in all of the six organs examined (liver, spleen, lung, kidney, bone marrow and colon). The mutant frequencies obtained with chrysene showed only small differences between the organs examined and ranged from 1.5 to 3 times the spontaneous frequency. The 4,10-DAC was more mutagenic than chrysene in all the organs tested. The highest lacZ mutation frequency was observed in the lung of 4,10-DAC-treated mice and it was 19 and 6 times the spontaneous frequency and the frequency induced by chrysene, respectively. The 1,10-DAC induced lacZ mutation in the lung with a frequency 4.3- and 1.5-fold higher than in the control and chrysene-treated mice, respectively, although the mutant frequencies in the other organs of 1,10-DAC-treated mice were almost equivalent to those of chrysene-treated mice. Not only chrysene but also DACs depressed the G:C to A:T transition and increased the G:C to T:A transversion in the liver and lung. These results suggest that the two types of nitrogen substitutions in the chrysene structure may enhance mutagenicity in the mouse lung, although they showed no difference in the target-organ specificity and the mutation spectrum.     

Dinitropyrenes induce gene mutations in multiple organs of the lambda/lacZ transgenic mouse (Muta Mouse)

Dinitropyrenes (DNPs), 1,3-, 1,6- and 1,8-dinitropyrene, are carcinogenic compounds found in diesel engine exhaust. DNPs are strongly mutagenic in the bacterial mutation assay (Ames test), mainly inducing frameshift type mutations. To assess mutagenicity of DNPs in vivo is important in evaluating their possible involvement in diesel exhaust-induced carcinogenesis in human. For this purpose, we used the lambda/lacZ transgenic mouse (Muta Mouse) to examine induction of mutations in multiple organs. A commercially available mixture of DNPs (1,3-, 1,6-, 1,8-, and unidentified isomer (s) with a content of 20.2, 30.4, 35.2, and 14.2%, respectively) was injected intragastrically at 200 and 400mg/kg once each week for 4 weeks. Seven days after the final treatment, liver, lung, colon, stomach, and bone marrow were collected for mutation analysis. The target transgene was recovered by the lambda packaging method and mutation of lacZ gene was analyzed by a positive selection with galE(-) E. coli. In order to determine the sequence alterations by DNPs, the mutagenicity of the lambda cII gene was also examined by the positive selection with hfl(-) E. coli. Since cII gene (294bp) is much smaller than the lacZ (3024bp), it facilitated the sequence analysis. Strongest increases in mutant frequencies (MFs) were observed in colon for both lacZ (7.5x10(-5) to 43.3x10(-5)) and cII (2.7x10(-5) to 22.5x10(-5)) gene. Three-four-fold increases were observed in stomach for both genes. A statistically significant increase in MFs was also evident in liver and lung for the lacZ gene, and in lung and bone marrow for the cII gene. The sequence alterations of the cII gene recovered from 37 mutants in the colon were compared with 50 mutants from untreated mice. Base substitution mutations predominated for both untreated (91%) and DNP-treated (84%) groups. The DNPs treatment increased the incidence of G:C to T:A transversion (2-43%) and decreased G:C to A:T transitions (70-22%). The G:C to T:A transversions, characteristic to DNPs treatment, is probably caused by the guanine-C8 adduct, which is known as a major DNA-adduct induced by DNPs, through an incorporation of adenine opposite the adduct ("A"-rule). The present study showed a relevant use of the cII gene as an additional target for mutagenesis in the Muta Mouse and revealed a mutagenic specificity of DNPs in vivo.     

In vivo genotoxicity evaluation of dimethylarsinic acid in Muta™Mouse

Dimethylarsinic acid (DMA) induces DNA damage in the lung by formation of various peroxyl radical species. The present study was conducted to evaluate whether arsenite or its metabolite, DMA, could initiate carcinogenesis via mutagenic DNA lesions in vivo that can be attributed to oxidative damage. A transgenic mouse model, Muta™Mouse, was used in this study and mutations in the lacZ transgene and in the endogenous cII gene were assessed. When DMA was intraperitoneally injected into Muta™Mice at a dose of 10.6 mg/kg per day for 5 consecutive days, it caused only a weak increase in the mutant frequency (MF) of the lacZ gene in the lung, which was at most 1.3-fold higher than in the untreated control animals. DMA did not appreciably raise the MF in the bladder or bone marrow. Further analysis of the cII gene in the lung, the organ in which DMA induced the DNA damage, revealed only a marginal increase in the MF. Following DMA administration, no change in the cII mutation spectra was observed, except for a slight increase in the G:C to T:A transversion. Administration of arsenic trioxide (arsenite) at a dose of 7.6 mg/kg per day did not result in any increase in the MF of the lacZ gene in the lung, kidney, bone marrow, or bladder. Micronucleus formation was also evaluated in peripheral blood reticulocytes (RETs). The assay for micronuclei gave marginally positive results with arsenite, but not with DMA. These results suggest that the mutagenicity of DMA and arsenite might be too low to be detected in the Muta™Mouse.     

In vivo genotoxicity of heterocyclic amines detected by a modified alkaline single cell gel electrophoresis assay in a multiple organ study in the mouse

We used a modification of the alkaline single cell gel electrophoresis (SCG) (Comet) assay to test the in vivo genotoxicity of 6 heterocyclic amines, Trp-P-1 (25 mg/kg), Trp-P-2 (13 mg/kg), IQ (13 mg/kg), MeIQ (13 mg/kg), MeIQx (13 mg/kg) and PhIP (40 mg/kg), in mouse liver, lung, kidney, brain, spleen, bone marrow and stomach mucosa. Mice were sacrificed 1, 3, and 24 h after intraperitoneal injection. Trp-P-2, IQ, MeIQ, and MeIQx yielded statistically significant DNA damage in the stomach, liver, kidney, lung and brain; Trp-P-1 in the stomach, liver and lung; and PhIP in the liver, kidney and brain. None of the heterocyclic amines induced DNA damage in the spleen and bone marrow. Our results suggest that the alkaline SCG assay applied to multiple organs is a good way to detect organ-specific genotoxicity of heterocyclic amines in mammals.     

Induction of lacZ mutation by 7,12-dimethylbenz[a]anthracene in various tissues of transgenic mice.

The induction of gene mutations was examined in MutaMouse after an intraperitoneal injection of 7, 8-dimethylbenz[a]anthracene (DMBA) at 20 mg/kg in a collaborative study participated by four laboratories. Although the DMBA dose used was lower than the level that has been reported to induce micronucleated erythrocytes maximally in several mouse strains, a killing effect appeared after day 9 of the post-treatment interval. Mutations in lacZ transgene were detected by the positive selection assay following in vitro packaging of phage lambda from the genomic DNA of the transgenic animals that survived. The mutant induction was evaluated in the bone marrow, liver, skin, colon, kidney, thymus, and testis 7 to 28 days after the treatment. In the bone marrow, the mutant frequency reached a maximum, approximately a 30-fold increase, 14 days after the treatment and the increased frequency persisted at least up to day 28 of the post-treatment. Induction of mutants was detected in the liver, colon, thymus, and skin to lesser extents. Marginal responses were obtained in the kidney and testis. The slight increases in the mutant frequencies in the kidney and testis observed in some laboratories were within laboratory-to-laboratory or animal-to-animal variations. In contrast to the gene mutation induction in the bone marrow, the frequency of micronucleated reticulocytes increased transiently 3 days after the treatment and returned to a control level before day 8 of the post-treatment. It was suggested that DMBA induced gene mutation is fixed in stem cells depending on cell proliferation while DNA damages responsible for chromosome breakage are not transmitted to progeny cells.     

Comparison of the mutational spectra of the lacZ transgene in four organs of the MutaMouse treated with benzo[a]pyrene: target organ specificity

We recently demonstrated that not all organs with a high rate of induction of mutation in the lacZ transgene develop tumors in the lambdalacZ transgenic mice (MutaMouse) used for a long-term carcinogenicity study with benzo[a]pyrene (BP). To better understand the role of chemical-induced in vivo mutations in carcinogenesis, we compared the mutational spectra of the lacZ transgene in four organs of the MutaMouse obtained 2 weeks after five daily consecutive oral treatments with 125 mg/kg/day BP. lacZ transgenes were analyzed in two target organs (forestomach and spleen) and two non-target organs (colon and glandular stomach) for BP-induced carcinogenesis in MutaMouse, and all of these organs were highly mutated in the lacZ transgene. The sequence data showed similar mutational spectra of the lacZ transgene between the two target organs; the predominant mutations were G:C-->T:A transversions (55% and 50% for forestomach and spleen, respectively), followed by deletions (20% and 21% for forestomach and spleen, respectively) mainly at G:C site. The frequent G:C-->T:A transversions are consistent with reports of the mutational spectra produced in the p53 gene in tumors generated in rats and mice exposed to BP. In contrast, the mutational spectra of the lacZ transgene in the two non-target organs are different from those in the target organs, and are also suggested to differ from one another. These findings suggest an organ/tissue-specific mechanism of mutagenesis.     

A lacZ transgenic mouse assay for the detection of mutations in follicular granulosa cells

There is ongoing concern that an assay for germ cell effects in female animals is not available. While transgenic mutation detection systems provide unprecedented access to numerous rodent tissues, studies on the induction of gene mutations in oocytes are still not possible because sufficient numbers of cells cannot be harvested. However, following stimulation of an ovarian follicle, the granulosa cells contained therein divide rapidly, increasing substantially in numbers. Since these granulosa cells share the same environment as the ovum, they may serve as suitable surrogates for the study of exposure of female germ cells to mutagens. Female lacZ transgenic mice (MutaMouse) were treated by intraperitoneal injection of N-ethylnitrosourea (ENU) and subsequently with pregnant mare serum gonadotropin (PMSG, 5IU/animal, i.p.) to induce follicular growth. Animals were sacrificed 48 h after the administration of PMSG and granulosa cells and bone marrow were harvested. A comparable dose-related increase in the mutant frequency (MF) of both granulosa and bone marrow cells was observed. The highest dose caused a decrease in the MF of granulosa cells, but not in the bone marrow, suggesting possible greater susceptibility of granulosa cells to ENU toxicity. Doubling dose estimates for bone marrow and granulosa cells were lower than those derived from the literature on oocyte mutation frequency using the Russell specific locus assay, suggesting that both cell types are more sensitive to ENU-induced mutation than oocytes. The results indicate that transgene mutations in granulosa cells may provide a sensitive pre-screening tool for potential genotoxic germ cell effects of exposed oocytes.     

Induction of gene mutations by 5-(2-chloroethyl)-2'-deoxyuridine (CEDU), an antiviral pyrimidine nucleoside analogue

5-(2-chloroethyl)-2'-deoxyuridine (CEDU) had been developed for the treatment of herpes simplex infections. In the Salmonella reverse mutation test, the compound was found to be mutagenic in strains TA1535 and TA102 at very high concentrations (> or =2500 micro g/plate), both with and without S9-mix. The mutagenic potential of CEDU was further investigated in vivo and in vitro. It did not induce DNA repair in rat hepatocyte primary cultures, and was negative in the micronucleus test in V79 cells and in the comet assay in human leukocytes. In vivo, CEDU was negative in the bone marrow micronucleus test in CD1 mice. The mouse spot test provided a clearly positive result. Treatment of mice on day 9 of pregnancy with 2000 mg/kg resulted in 5.9% of the F1 animals having genetically relevant spots, whereas the corresponding vehicle control group had a spot rate of 1.9%. Since these data clearly identified CEDU as an inducer of gene mutations in vivo, this potential was further investigated in lacZ transgenic Muta Mouse. Six female animals were treated daily on five consecutive days with 2000 mg/kg/day and sacrificed, after a treatment-free sampling time, 14 days later. The data showed a clear increase in the mutant frequency in the bone marrow, the lung and in the spleen. CEDU is an exception in the group of nucleoside analogues, because it was found to be a strong gene mutagen and, in contrast to the other compounds of this group investigated so far, had no considerable clastogenic effects.     

Induction of A:T to G:C transition mutations by 5-(2-chloroethyl)-2'-deoxyuridine (CEDU), an antiviral pyrimidine nucleoside analogue, in the bone marrow of Muta Mouse

5-(2-chloroethyl)-2'-deoxyuridine (CEDU) is a pyrimidine nucleoside analogue formerly in development for the treatment of herpes simplex virus infections. The compound proved clearly mutagenic in the mouse spot test and exhibited weak activity in the Salmonella reverse mutation test, which led to the termination of the compound's development. In another study, CEDU, administered orally to beta-galactosidase (lacZ) transgenic mice (Muta Mouse) for five days, induced a clear increase in lacZ mutant frequencies in spleen, lung, and bone marrow. In the present follow-up study, we analyzed 32 of those lacZ mutants isolated from the bone marrow of the Muta Mouse animals of the experiments mentioned above, in order to obtain further information on the type of mutations induced by CEDU. CEDU induced a pronounced increase in A:T to G:C transitions. The distribution of A:T to G:C transitions was clearly non-random, showing a bias towards T to C substitutions in the coding DNA strand and a preference to occur in the sequence motif 5'-(G or C)-T-G-3'. Our data support the hypothesis that CEDU, after being phosphorylated, is incorporated into cellular DNA in place of thymidine, which leads to mispairing with guanosine during subsequent DNA replication. As a result, the compound is thought to exert its mutagenicity by inducing mismatches leading to T to C transitions. Our findings point towards a mode of mutagenic action of CEDU that differs fundamentally from that of other antiviral antinucleosides whose clastogenic and recombinogenic activities prevail.     

The enhancer domain of the human cytomegalovirus major immediate-early promoter determines cell type-specific expression in transgenic mice.

The human cytomegalovirus (HCMV) major immediate-early promoter (MIEP) is one of the first promoters to activate upon infection. To examine HCMV MIEP tissue-specific expression, transgenic mice were established containing the lacZ gene regulated by the MIEP (nucleotides -670 to +54). In the transgenic mice, lacZ expression was demonstrated in 19 of 29 tissues tested by histochemical and immunochemical analyses. These tissues included brain, eye, spinal cord, esophagus, stomach, pancreas, kidney, bladder, testis, ovary, spleen, salivary gland, thymus, bone marrow, skin, cartilage, and cardiac, striated and smooth muscles. Although expression was observed in multiple organs, promoter activity was restricted to specific cell types. The cell types which demonstrated HCMV MIEP expression included retinal cells of the eye, ductile cells of the salivary gland, exocrine cells of the pancreas, mucosal cells of the stomach and intestine, neuronal cells of the brain, muscle fibers, thecal cells of the corpus luteum, and Leydig and sperm cells of the testis. These observations indicate that the HCMV MIEP is not a pan-specific promoter and that the majority of expressing tissues correlate with tissues naturally infected by the virus in the human host.     

Mutagenicity of the peroxisome proliferators clofibrate, Wyeth 14,643 and di-2-ethylhexyl phthalate in the lacZ plasmid-based transgenic mouse mutation assay

BACKGROUND: Peroxisome proliferators are considered rodent carcinogens that are putative human non-carcinogens based on the presumed absence of direct genetic toxicity in rodent and human cells and the resistance of human cells to the induction of peroxisomes by peroxisome proliferators. The highly sensitive lacZ plasmid-based transgenic mouse mutation assay was employed to investigate the mutagenicity of several peroxisome proliferators based on several lines of evidence suggesting that these agents may in fact exert a genotoxic effect. METHODS: Male and female lacZ-plasmid based transgenic mice were treated at 4 months of age with 6 doses of 2,333 mg di-2-ethylhexyl phthalate (DHEP), 200 mg Wyeth-14,643, or 90 mg clofibrate per kg of bodyweight, respectively, over a two-week period. Control animals were treated with the respective vehicles only (35% propyl glycol for DEHP and Wyeth-14,643 treatment controls and sterile water for clofibrate treatment controls).The mutant frequency in liver, kidney and spleen DNA was determined as the proportion of retrieved mutant and wild-type lacZ plasmids expressed in Escherichia Coli C host cells employing a positive selection system for mutant plasmids. RESULTS: Exposure to DEHP or Wyeth-14,643 significantly increased the mutant frequency in liver, but not in kidney or spleen, of both female and male mice. Treatment with clofibrate did not lead to an increased mutant frequency in any of the organs studied. CONCLUSION: The results indicate that some peroxisome proliferators display an organ-specific mutagenicity in lacZ plasmid-based transgenic mice consistent with historical observations of organ- and compound-specific carcinogenicity.     

Mutagenicity of aristolochic acid in the lambda/lacZ transgenic mouse (MutaMouse)

Aristolochic acid (AA) is found in a plant that causes urothelial carcinomas in patients with Chinese herb nephropathy (CHN). To evaluate the in vivo mutagenicity of AA, we analysed the mutant frequency (MF) in the lacZ and cII gene of 10 organs of the lambda/lacZ transgenic mouse (MutaMouse) after intragastric treatment with AA (15mg/kg per week x 4). Simultaneously, the clastogenicity of AA was evaluated by the peripheral blood micronucleus assay. The nature of the mutations induced by AA was revealed by the sequence analysis of the cII gene, which is also a phenotypically selectable marker in the lambda transgene. MFs in the target organs-forestomach, kidney, and bladder of AA-treated mice were significantly higher than those of control mice (forestomach 33- and 15-fold; kidney 10- and 9-fold; bladder 16- and 31-fold, for the lacZ and cII, respectively). The MFs in non-target organs, except the colon, showed only slight increases. Sequence analysis of cII mutants in target organs revealed that AA induced mainly A:T to T:A transversions whereas G:C to A:T transitions at CpG sites predominated among spontaneous mutations. These results suggested that AA, which is activated by cytochrome P450 and peroxidase to form cyclic nitrenium ions that bind to deoxyadenine, caused the A to T transversions in the target organs of mice.