Tissue-specific mutant frequencies and mutational spectra in cyclophosphamide-treated lacI transgenic mice.

The induction and nature of mutations in the lacI transgene were evaluated in multiple tissues after exposure of adult male B6C3F1 lacI transgenic mice to cyclophosphamide (CP). Mice were given a single i.p. injection of 25 mg CP/kg, 100 mg CP/kg, or vehicle (PBS) and then necropsied 6 weeks after treatment to allow DNA extraction and lacI mutant recovery. Tissues evaluated included target tissues for tumorigenesis (lung, urinary bladder) and sites not susceptible to tumor formation in B6C3F1 mice (kidney, bone marrow, splenic T-lymphocytes). After exposure to the high dose of CP, a significant increase in the mutant frequency (Mf) was detected in the lungs and urinary bladders, compared to the respective tissues from vehicle-treated controls. In contrast, the Mfs in kidney, bone marrow, and splenic T cells from CP-treated mice were not significantly different from controls. The spectra of mutations in lacI from lung and urinary bladder were significantly changed after high-dose CP treatment, with a significant increase in the frequency of A. T --> T. A transversions found in both tissues and a significantly elevated frequency of deletions in the lungs. Conversely, in vehicle-treated mice, the two predominant classes of lacI mutations recovered in lung and urinary bladder were G. C --> A. T transitions at CpG sites and G. C --> T. A transversions. These CP exposures were also genotoxic as measured by the significant induction of micronuclei in peripheral blood 48 hr after exposure. These data indicate that under these study conditions, CP-induced mutations are detectable in the lacI transgene in the target tissues, but not in nontarget tissues for CP-induced cancer. With the lacI assay it is possible to study mutagenicity in a variety of critical tissues to provide mechanistic information related to genotoxicity and carcinogenicity in vivo.     

Hprt mutant frequencies in splenic T-cells of male F344 rats exposed by inhalation to propylene

Propylene is a major industrial intermediate and atmospheric pollutant to which humans are exposed by inhalation. In this study, 6-week-old male F344 rates were exposed to 0, 200, 2000, or 10,000 ppm propylene by inhalation for 4 weeks (6 h/day, 5 days/week), and mutant frequencies were determined in the Hprt gene of splenic T-lymphocytes. Twenty milligrams of cyclophosphamide monohydrate (CPP)/kg bw, given on the penultimate day of propylene exposure, was used as a positive control mutagen. Rats (n = 8/group) were necropsied for isolation of T-cells 8 weeks after the last dose, a sampling time that produced peak spleen Hprt mutant frequencies (Mfs) in a preliminary mutant manifestation study using CCP treatment. Hprt Mfs were measured via the T-cell cloning assay, which was performed without knowledge of the animal treatment groups. Mean Hprt Mfs were significantly increased over control values (mean Mf = 5.24 +/- 1.55 (SD) x 10(-6)) in CPP-treated rats (10.37 +/- 4.30 x 10(-6), P = 0.007). However, Hprt Mfs in propylene-exposed rats were not significantly increased over background, with mean Mfs of 4.90 +/- 1.84 x 10(-6) (P = 0.152), 5.05 +/- 3.70 x 10(-6) (P = 0.895), and 5.95 +/- 2.49 x10(-6) (P = 0.500) for animals exposed to 200, 2000, or 10,000 ppm propylene, respectively. No significant increase in F344 rat or B6C3F1 mouse cancer incidence was reported in the National Toxicology Program carcinogenicity studies of propylene across this same exposure range. Taken together, these findings support the conclusion that inhalation exposure of rats to propylene does not cause mutations or cancer.     

Survival and tumorigenesis in O6-methylguanine DNA methyltransferase-deficient mice following cyclophosphamide exposure

O(6)-methylguanine DNA methyltransferase (MGMT) deficiency is associated with an increased susceptibility to alkylating agent toxicity. To understand the contribution of MGMT in protecting against cyclophosphamide (CP)-induced toxicity, mutagenesis and tumorigenesis, we compared the biological effects of this agent in transgenic Mgmt knockout and wild-type mice. In addition, neurofibromin (Nf1)+/- background was used to increase the likelihood of CP-induced tumorigenesis. Cohorts of Mgmt-proficient or -deficient mice (either Nf1+/+ or Nf1+/-) were given 6 weekly injections of a maximally tolerated dose of CP (250 mg/kg) or vehicle and followed for 15 months. CP-treated mice had more deaths than control mice but there was no difference in the long-term survival between Mgmt+/+ and Mgmt-/- mice (12 of 83 Mgmt+/+ mice died compared to 12 of 80 Mgmt-/- mice, disregarding Nf1 status). Lymphomas and adrenal tumours were the most frequent malignancies. Interestingly, CP-treated, Mgmt-deficient mice developed fewer tumours than controls. Ten of 71 (14%) Mgmt-proficient mice developed tumours after CP treatment compared to only 2 of 68 (3%) Mgmt-deficient mice (P = 0.02). Mgmt-/-, Nf1+/- mice developed fewer tumours (1 of 35, 3%) following CP compared to Mgmt+/+, Nf1+/- mice (7 of 37, 19%) (P = 0.03). Hypoxanthine-guanine phosphoribosyltransferase mutation assays showed no significant increases in mutant frequencies in Mgmt-/- (18.1 x 10(6)) compared to Mgmt+/+ mice (12.9 x 10(6)). These data indicate that MGMT deficiency does not protect against long-term toxicity or mutagenicity from CP and appears to attenuate the occurrence of CP-induced tumours in an Nf1+/- background.     

Transplacental mutagenicity of N-ethyl-N-nitrosourea at the hprt locus in T-lymphocytes of exposed B6C3F1 mice

Previous studies have compared age-related differences in total mutagenic burden in mice of differing age (preweanling, weanling, or young adult) after single intraperitoneal (i.p.) injections of ethylnitrosourea (ENU). The purpose of the present investigation was to determine the effects of time elapsed since treatment on the frequency of hprt mutant T-cells (Mf) from mice treated transplacentally with single acute vs. multiple split doses of ENU. To this end, pregnant C57BL/6 mice (n = 13-16/group), which had been bred to C3H males, were given i.p. injections of 40 mg ENU/kg bw in a single dose on day 18 of gestation, in a split dose of 6 mg ENU/kg bw on days 12 through 18 of gestation, or DMSO vehicle alone. Groups of pups were necropsied on days 10, 13, 15 (single dose only), 17, 20, 40, and 70 postpartum for T-cell isolations and hprt Mf measurements using the T-cell cloning assay. The time required to reach maximum Mfs in T-cells isolated from thymus of transplacentally treated animals was 2 weeks, the same time span as previously observed after ENU treatment of adult, weanling, and preweanling mice. Mfs in T-cells isolated from spleens of control animals averaged 2.1 +/- 0.3 (SE) x 10(-6). In spleens of mice treated transplacentally with ENU in a single dose, Mfs reached a maximum at 15 days postpartum [84.7 +/- 15.8 (SE) x 10(-6)] and decreased to lower but still elevated levels at 40 days postpartum. In spleens of mice treated transplacentally with ENU in a split dose, Mfs reached a maximum at 13 days postpartum [74.0 +/- 16.3 (SE) x 10(-6)] and decreased to background levels at 40 days postpartum. The areas under the curves describing the change in hprt Mfs over time for ENU-treated vs. control mice estimate the mutagenic potency for transplacental single- and split-dose exposures to be 1.9 and 0.8 x 10(3), respectively. Comparison of the mutagenic potency estimates for mice exposed to ENU in utero to 4-week-old mice given a similar dose of the same lot number of ENU indicates that the mouse is more susceptible to ENU-induced mutagenesis during fetal life.     

Effect of chronic azathioprine treatment on germ-line transmission of Hprt mutation in mice

Azathioprine (Aza), a prodrug of 6-mercaptopurine, is used in human medicine to prevent transplant rejection and for the treatment of autoimmune diseases. Extremely high HPRT lymphocyte mutant frequencies (MFs) are found in humans and mice chronically treated with Aza, and these elevated MFs appear to be caused by selection and amplification of pre-existing HPRT mutant lymphocytes. In the present study, we investigated if in vivo selection by Aza also promotes the germ-line transmission of Hprt mutants. Fifty-five male C57BL/6 mice were treated with 10 mg/kg Aza three times/week for 24 weeks; 10 control mice were treated with the vehicle. Each of these males then was bred to unexposed females for a total of 8 weeks. Analysis of the Aza-treated males after the breeding period indicated that 12 had highly elevated Hprt lymphocyte MFs (1 x 10(-4)-2.5 x 10(-1) vs. normal MFs of <1 x 10(-5)), indicating that the Aza treatment successfully selected somatic cell mutants. The female offspring from the breeding were sacrificed at 28 days of age and Hprt MFs were measured in spleen lymphocytes. Most of the 364 female offspring (332 from Aza-treated fathers) had Hprt MFs of 0-6 x 10(-6), but seven of the offspring had moderately elevated MFs of 16 x 10(-6)-55 x 10(-6). Since one of these mice was fathered by a control male, these relatively high MFs appear to be part of the normal variation in lymphocyte Hprt MF. The present results provide no evidence that long-term Aza treatment promotes high levels of germ-line Hprt mutation transmission in mice.     

Comparison of mutant frequencies at the transgenic lambda LacI and cII/cI loci in control and ENU-treated Big Blue mice.

We compared the lambda cII/cI transgenic mutation assay described by Jakubczak et al. [(1996): Proc Natl Acad Sci USA 93:9073-9078] to the previously established Big Blue assay. Genomic DNA isolated from liver, spleen, and lung tissue of control or ethylnitrosourea (ENU)-treated Big Blue mice (100 mg/kg i.p., single dose) was packaged into phage (five animals, two packagings per DNA sample) which were simultaneously plated for lacI and cII/cI mutant frequency (MF) and titer. Mean MF of control animals was higher for cII/cI than lacI for all three tissues examined (spontaneous cII/cI MF divided by spontaneous lacI MF = 2.9, 3.1, and 1.7 for liver, spleen, and lung, respectively). The differences were statistically significant for liver and spleen, but not lung. The ENU-induced MF measured by subtracting control MFs from ENU-treated MFs was higher in the cII/cI assay than lacI (liver = 23.0 x 10(-5) for cII/cI vs. 15.1 x 10(-5) for lacI; spleen = 64.8 x 10(-5) for cII/cI vs. 36.1 x 10(-5) for lacI; lung = 17.1 x 10(-5) for cII/cI vs. 15.8 x 10(-5) for lacI). Fold increase over control values measured by dividing MF of ENU-treated animals by appropriate control values was higher for lacI than cII/cI (liver = 4.4-fold for lacI vs. 2.7 for cII/cI; spleen = 13.1-fold for lacI vs. 8.4 for cII/cI; and lung = 5.6-fold for lacI vs. 4.0 for cII/cI). Despite these differences, overall results were similar for the two mutational endpoints. These results suggest that the cII/cI assay may be an acceptable alternative to lacI where transgenic mutation studies are indicated.     

Transmission of mutations in the lacI transgene to the offspring of ENU-treated Big Blue male mice

The purpose of the study was to determine: 1) if male germ cells of Big Blue mice carrying newly induced mutations in the lacI transgene were effective in fertilization; 2) if offspring arising from such mutant sperm had the mutation in germ cells and multiple somatic tissues; and 3) how the frequency of mutants induced in the lacI transgene compared to the frequency induced in endogenous genes traditionally employed to study germ cell mutagenesis in mice. Male B6C3F(1) mice hemizygous for the lambda/lacI transgene were treated weekly with 100 mg/kg body weight of the mammalian germ cell mutagen N-ethyl-N-nitrosourea (ENU). The cumulative dose for each treated animal was 300 mg ENU/kg body weight. Ten weeks later the treated mice were mated to T stock females and the resulting offspring were screened for specific-locus mutations at six loci affecting external appearance, as well as for mutations in the lacI transgene in multiple somatic tissues and germ cells. Five offspring carrying recessive specific-locus mutations were observed among 597 offspring screened (mutant frequency = 139.6 x 10(-5) per locus). Four offspring carrying lacI mutations were observed among 280 offspring screened (mutant frequency = 35.7 x 10(-5) per locus (assuming 40 target loci)). Each of the four lacI mutant offspring carried a different mutation. Three of the mutations were A:T-->G:C transitions and one a G:C-->A:T transition. Consistent with the expectation that a mutation induced in a parental germ cell and transmitted to a conceptus would exist in every cell of the offspring, each mutant mouse had identical mutations in all somatic tissues sampled, as well as in its germ cells. These data provide preliminary evidence for the biological validity of assessing induced, heritable mutations using transgenic mice, without the need for generating an F(1) generation.     

Comparison of hprt and lacI mutant frequency with DNA adduct formation in N-hydroxy-2-acetylaminofluorene-treated Big Blue rats

N-Hydroxy-2-acetylaminofluorene (N-OH-AAF) is the proximate carcinogenic metabolite of the powerful rat liver carcinogen 2-acetylaminofluorene. In this study, transgenic Big Blue(R) rats were used to examine the relationship between in vivo mutagenicity and DNA adduct formation by N-OH-AAF in the target liver compared with that in nontarget tissues. Male rats were given one, two, or four doses of 25 mg N-OH-AAF/kg body weight by i.p. injection at 4-day intervals, and groups of treated and control rats were euthanized up to 10 weeks after beginning the dosing. Mutant frequencies were measured in the spleen lymphocyte hprt gene, and lacI mutant frequencies were determined in the liver and spleen lymphocytes. At 6 weeks after beginning the dosing, the hprt mutant frequency in spleen lymphocytes from the four-dose group was 16.5 x 10(-6) compared with 3.2 x 10(-6) in control animals. Also at 6 weeks, rats given one, two, or four doses of N-OH-AAF had lacI mutant frequencies in the liver of 97.6, 155.6, and 406.8 x 10(-6), respectively, compared with a control frequency of 25.7 x 10(-6); rats given four doses had lacI mutant frequencies in spleen lymphocytes of 55.8 x 10(-6) compared with a control frequency of 20.4 x 10(-6). Additional rats were evaluated for DNA adduct formation in the liver, spleen lymphocytes, and bone marrow by (32)P-postlabeling. Adduct analysis was conducted 1 day after one, two, and four treatments with N-OH-AAF, 5 days after one treatment, and 9 days after two treatments. N-(Deoxyguanosin-8-yl)-2-aminofluorene was the major DNA adduct identified in all the tissues examined. Adduct concentrations increased with total dose to maximum values in samples taken 1 day after two doses, and remained essentially the same after four doses. In samples taken after four doses, adduct levels were 103, 28, and 7 fmol/microg of DNA in liver, spleen lymphocytes, and bone marrow, respectively. The results indicate that the extent of both DNA adduct formation and mutant induction correlates with the organ specificity for N-OH-AAF carcinogenesis in the rat. Environ. Mol. Mutagen. 37:195-202, 2001. Published 2001 Wiley-Liss, Inc.     

Frequency and spectrum of ENU-induced mutation in the Phi X174 transgene in mouse splenic lymphocytes and their significance to spontaneous transgenic rodent mutation frequencies

A perceived disadvantage of transgenic rodent mutation assays is that spontaneous mutant frequencies are high compared to those of endogenous genes and may consequently reduce sensitivity to induced mutation. We have previously argued that unrepaired G:T mismatches from spontaneous deamination of 5-methylcytosine at CpG sites could be converted to apparent in vivo mutations in the bacterial recovery systems because of rapid, random, mismatch repair in Escherichia coli. In this study, we have measured mutation frequencies in spleen of male mice induced by N-ethyl-N-nitrosourea (ENU) using the PhiX174 transgene, which is not subject to mismatch repair in E.coli, using single-burst analysis, a unique method to identify in vivo mutation. In order to compare our results to those using the lacI and cII transgenes, we converted all mutant frequencies to base pair substitution (bps) mutation frequencies per nucleotide based on mutant spectra from this study and published literature. We found this frequency in control spleen to be similar for lacI (3.8 +/- 0.7 x 10(-8)) and PhiX174 (3.1 +/- 1.2 x 10(-8)) at 6 weeks of age. We found a strong age dependence for spontaneous lacI mutation that extrapolated to a value at conception (1.8 +/- 0.9 x 10(-8)) that was not significantly different from the human germ line bps mutation frequency per nucleotide of 1.7 +/- 0.2 x 10(-8). These two transgenes provided similar mutational responses to 40 mg/kg ENU, 7- to 9-fold. In contrast, the cII target gene in the same tissue produces both spontaneous and induced mutation frequencies approximately 10 times higher, for unknown reasons. We conclude that the spontaneous mutant frequencies measured by the lacI and PhiX174 transgenes in this moderately dividing tissue accurately measure in vivo mutation frequencies at early ages. For these two transgenes, seemingly high mutant frequencies may reflect the expected accumulation of somatic mutation with age.     

Somatic mutant frequency at the HPRT locus in children associated with a pediatric cancer cluster linked to exposure to two superfund sites

The somatic mutant frequency (Mf) of the hypoxanthine phosphoribosyl transferase (HPRT) gene has been widely used as a biomarker for the genotoxic effects of exposure but few studies have found an association with environmental exposures. We measured background Mfs in 49 current and former residents of Dover Township, New Jersey, who were exposed during childhood to industrially contaminated drinking water. The exposed subjects were the siblings of children who developed cancer after residing in Dover Township, where the incidence of childhood cancer has been elevated since 1979. Mfs from this exposed group were compared to Mfs in 43 age-matched, presumably unexposed residents of neighboring communities with no known water contamination and no increased cancer incidence. Statistical comparisons were based on the natural logarithm of Mf (lnMF). The mean Mf for the exposed group did not differ significantly from the unexposed group (3.90 x 10(-6) vs. 5.06 x 10(-6); P = 0.135), but unselected cloning efficiencies were higher in the exposed group (0.55 vs. 0.45; P = 0.005). After adjustment for cloning efficiency, lnMf values were very similar in both groups and age-related increases were comparable to those previously observed in healthy children. The results suggest that HPRT Mf may not be a sensitive biomarker for the genotoxic effects of environmental exposures in children, particularly when substantial time has elapsed since exposure.     

Mutant frequency and molecular analysis of in vivo lacI mutations in the bone marrow of Big Blue rats treated with 7, 12-dimethylbenz[a]anthracene

Recently, we evaluated lacI mutations in lymphocytes and mammary tissue of Big Blue (BB) rats exposed to 7, 12-dimethylbenz[a]anthracene (DMBA). The results on the time course of mutant induction suggested that the lacI gene may manifest a tissue-specific increase in mutant frequency (MF). To test whether a tissue-specific increase in lacI MF is dependent on the cell proliferation rate of a tissue, we examined rapidly proliferating bone marrow cells for DMBA-induced lacI mutations. Seven-week-old female BB rats were given single doses of 0, 20, and 130 mg/kg DMBA by gavage and the lacI MFs in the bone marrow were measured over a period of 14 weeks following treatment. Bone marrow cells had a remarkably low average background MF (3.1 +/- 1.6 x 10(-6) plaque-forming units) and the DMBA-induced lacI MFs were significantly higher than control MFs for both doses and at all time points (P < 0.01). The lacI MF in the bone marrow increased for 2 weeks and then remained relatively constant; 20 and 130 mg/kg DMBA produced 34- and 106-fold increases in MF over control MF. DNA sequencing revealed that the majority of DMBA-induced lacI mutations were base-pair substitutions and that A:T --> T:A (48%) and G:C --> T:A (24%) transversions were the predominant types. Thus, the different lacI mutation fixation times observed for bone marrow (2 weeks), mammary (10 weeks), and lymphocytes (6 weeks) suggest that the lacI gene manifests a tissue-specific mutation fixation time, which may depend on the cell proliferation rate of a tissue. In addition, the relatively low spontaneous MF in bone marrow compared with that in other tissues may be useful for increasing the sensitivity of the assay for detecting induced MFs in BB rats.     

Comparative analysis of HPRT mutant frequency in children with cancer

The link between exposure to environmental mutagens and the development of cancer is well established. Yet there is a paucity of data on the relationship between gene-environment interactions and the mechanisms associated with the somatic mutational events involved with malignant transformation, especially in children. To gain insight into somatic mutational mechanisms in children who develop cancer, we determined the background mutant frequency (Mf) in the hypoxanthine phosphoribosyl transferase (HPRT) reporter gene of peripheral blood lymphocytes from pediatric cancer patients at the time of diagnosis and prior to therapeutic intervention. We studied 23 children with hematologic malignancies and 31 children with solid tumors prior to initial therapeutic intervention. Children with solid tumors, specifically sarcomas, and Hodgkin's disease were significantly older and had elevated HPRT Mfs (6.1 x 10(-6) and 3.7 x 10(-6), respectively) at the time of diagnosis, compared to normal controls (2.3 x 10(-6)) and other pediatric tumor groups including children with acute lymphocytic leukemia and non-Hodgkin's lymphoma (ALL/NHL, 1.7 x 10(-6)), central nervous system tumors (CNS, 3.6 x 10(-6)), and neuroblastoma (1.9 x 10(-6)). Of importance is that the significant differences observed in HPRT Mfs between these groups no longer existed after correcting for the effects of age. These data demonstrate that in children who develop cancer there appears to be no significant increase in background HPRT Mf that would indicate significant exposure to genotoxic chemicals or an underlying DNA repair defect resulting in genomic instability. In addition, these data demonstrate the importance of correcting for the effect of age when comparing the frequency of somatic mutations in children and should provide baseline data for future longitudinal biomonitoring studies on the genetic effects of chemotherapy in children treated for cancer.     

7,12-dimethylbenz[a]anthracene-induced mutation in the Tk gene of Tk(+/-) mice: automated scoring of lymphocyte clones using a fluorescent viability indicator

7,12-Dimethylbenz[a]anthracene (DMBA) is a rodent carcinogen and a potent in vivo mutagen for the X-linked hypoxanthine guanine phosphoribosyl transferase (hprt) gene of rats and for the lacI transgene of Big Blue mice and rats. Although DMBA is also a powerful clastogen, molecular analysis of these DMBA-induced hprt and lacI mutations indicates that most are single base-pair (bp) substitutions and 1- to 3-bp frameshifts. In the present study, we evaluated the types of mutations induced by DMBA in the autosomal thymidine kinase (Tk) gene of Tk(+/-) mice. Male and female 5- to 6-week-old animals were injected i.p. with DMBA at a dose of 30 mg/kg. Five weeks after the treatment, hprt and Tk mutant frequencies were determined using a limiting dilution clonal assay in 96-well plates. We established conditions for the automated identification of wells containing expanded lymphocyte clones using the fluorescent indicator alamarBlue. This procedure allowed the unbiased identification of viable clones and calculation of mutant frequencies. In male mice, DMBA treatment increased the frequency of hprt mutants from 1.8 +/- 1.1 to 34 +/- 9 x 10(-6), and Tk mutants from 33 +/- 12 to 78 +/- 26 x 10(-6); treated female mice had a significant but lower increase in hprt mutant frequency than did males. Molecular analysis of DMBA-induced Tk mutants revealed that at least 75% had the entire wild-type Tk allele missing. The results indicate that the predominant types of DMBA-induced mutation detected by the autosomal Tk gene are different from those detected by the X-linked hprt gene. The Tk gene mainly detects loss of heterozygosity mutation, whereas the majority of mutations previously found in the hprt gene were point mutations.     

Inability of cyclophosphamide-induced tolerance to permit engraftment of pluripotent stem cells contained in a moderate number of syngeneic bone marrow cells

We have previously described cyclophosphamide (CP)-induced tolerance that comprises an intravenous (i.v.) injection of 1 x 10(8) allogeneic spleen cells (SC) followed, 2 days later, by an intraperitoneal (i.p.) administration of 200 mg/kg of CP. By using this method, we were able to readily induce both long-lasting mixed chimerism and skin allograft tolerance in most of H-2 matched combinations, but not in H-2 mismatched combinations. In the present study, we have investigated whether the treatment with SC followed by 200 mg/kg CP can permit engraftment of syngeneic pluripotent hematopoietic stem cells (HSC), and whether CP itself can permit the HSC engraftment. Recipient B6 (H-2b; Ly-5.2) mice received 1 x 10(7), 5 x 10(7) or 1 x 10(8) SC from syngeneic B6.Ly-5.1 (H-2b; Ly-5.1) mice and administered 2 day later 200 mg/kg CP. Using anti-Ly-5.1 and Ly-5.2 mAbs and a flow cytometry, the origins of lymphoid and myeloid cells injecting the recipients were observed over time. Chimerism was at least initially detectable in all groups treated with SC alone (without 200 mg/kg CP), but became undetectable by 32 weeks after the treatment with SC alone. In recipient B6 mice treated with B6.Ly-5.1 SC and 200 mg/kg CP, on the other hand, lymphoid-chimerism was detectable at 32 weeks in a transferred cell dose-dependent manner, but granulocyte-chimerism indicating pluripotent HSC engraftment disappeared earlier than 32 weeks after the treatment. In order to further evaluate the effect of CP itself on HSC engraftment, recipient B6 mice were given various doses of CP (50-400 mg/kg) and were injected with T cell-depleted 1 x 10(7) BMC from B6.Ly-5.1 mice. Multilineage mixed chimerism over 32 weeks was induced in only one of 11 B6 mice treated with 400 mg/kg CP followed by B6.Ly-5.1 BMC, although lymphoid chimerism was induced temporarily in recipient B6 mice treated with 200 mg/kg CP followed by B6.Ly-5.1 BMC and persistently in most of recipient B6 mice treated with 400 mg/kg CP followed by B6.Ly-5.1 BMC.     

Benefit of Satureja khuzestanica in subchronically rat model of cyclophosphamide-induced hemorrhagic cystitis

Cyclophosphamide (CP) as a widely used antineoplastic drug causes hemorrhagic cystitis (HC) mainly via induction of oxidative stress. Regarding established antioxidant potential of Satureja khuzestanica (Lamiaceae) essential oil (SKEO), we aimed to investigate its protective effects in a subchronic rat model of CP-induced HC. CP (6 mg/kg/day) and SKEO (225 mg/kg/day) were administered alone or in combination by gavage for 28 days. Histopathological changes were investigated by light microscopy. Plasma samples were assayed for lipid peroxidation and total antioxidant power as biomarkers of toxic stress.In the CP-treated animals, irregular mucus layer, severe hemorrhage and edema, infiltration of inflammatory cells, and accumulation of mast cells were observed. In the CP+SKEO group, a relatively normal urothelial topography with decreased number of mucosal mast cells and inflammatory cells were observed. Increased lipid peroxidation along with decreased total antioxidant capacity resulting from CP treatment was significantly recovered by SKEO co-treatment.It is concluded that SKEO protects rats from CP-induced HC by reduction of free radical-induced toxic stress. It is strongly recommended to examine SKEO in the clinic to approve its benefit in patients undertaking CP.     

Transplacental teratogenesis and mutagenesis in mouse fetuses treated with cyclophosphamide

We studied transplacental fetotoxicity, teratogenicity, and mutagenicity in Swiss Webster mice following different doses of cyclophosphamide (CP; 0, 5, 10, 15, or 20 mg/kg), a well-known mutagen/teratogen, on day 12 of gestation. The fetal survival and weight on day 18 of gestation decreased significantly with increasing CP dose (P less than 0.01). The CP-treated fetuses were also dysmorphic (e.g., shortened limbs, digital defects, cleft palate, open eyes, and hydrocephaly) and the percentage of dysmorphology increased with increasing CP doses (P less than 0.01). To evaluate mutagenesis, a separate group of females received 5-bromodeoxyuridine tablet (50-mg) implants on day 12 of gestation and a CP treatment 8 h later. Fetal liver cells were harvested 24 h post-BrdU implant to analyze sister chromatid exchange (SCE) frequency and micronuclei. CP caused a significant increase in the SCEs per fetal liver cell from 3.4 +/- 0.02 (control) to 90.0 +/- 0.04 (20 mg/kg CP) (P less than 0.01). The increasing CP dose was also related to an increase in micronuclei. The data suggest that CP is transplacentally toxic, teratogenic, and mutagenic. Further analyses of the data suggest that the mutagenic effects of CP may in fact contribute indirectly to the CP-related teratogenic effects. Such conclusions are based on path analysis with directional causations associated with SCEs per cell and the dysmorphic features studied.     

Pharmacological validation of a model of cystitis pain in the mouse

Interstitial cystitis (IC) is a chronic pelvic-perineal pain syndrome of unknown etiology that mainly targets the lower urinary tract. Pain is the most prominent feature of IC and current therapies provide limited relief. Novel treatment options for IC could be identified if more predictive animal models were available. A rat model based on administration of cyclophosphamide (CP) mimics the symptoms of IC and has been well characterized. However, experiments in mice have not consistently reported both the spontaneous and evoked pain behaviors. The current series of studies demonstrate that CP (200-400mg, i.p.) increased both spontaneous and evoked pain behaviors in mice. Additionally, clinically relevant compounds: morphine (1-10mg/kg), ketorolac (1-5.6mg/kg) and duloxetine (3-30mg/kg) all significantly reversed pain behaviors. In contrast, gabapentin (56mg/kg) had no effect. Thus, CP-induced cystitis in mice may be used to evaluate novel therapeutics for the treatment of pain due to interstitial cystitis.     

The effects of cyclophosphamide, melatonin and carvedilol on neural tube and skeletal system of mice fetuses in prenatal period

Cyclophosphamide (CP) as an alkylating agent is used for treatment of cancer and to prevent rejection of tissue transplantation. There are many reports that the teratogenic effects of cyclophosphamide can be prevented by application of antioxidant drugs and stimulation of the maternal immune system. Also, there is some evidence that melatonin and carvedilol are antioxidant.Therefore, in this study, the prophylactic effects of melatonin and carvedilol on teratogenic effects of CP was compared. This study was performed on 31 pregnant mice that were divided into six groups. The control group received normal saline and test groups received CP (20 mg/kg), carvedilol (5 mg/kg), melatonin (10 mg/kg), CP (20 mg/kg) pluscarvedilol (5 mg/kg) and CP (20 mg/kg) plus melatonin (10 mg/kg) intraperitoneally on the 10th day of gestation, respectively. Fetuses were collected on the 19th day of gestation and after determination of weight and length; they were stained by Alizarin red-Alcian blue method. Cleft palate, spina bifida and exencephalyincidence were 62.79%, 62.79% and 30.23% in fetuses of mice that received only CP. Cleft palate,spina bifida, exencephaly, and incidence were 45.45%, 9.09% and 0% in group which received CP plus carvedilol (5 mg/kg), respectively.However, cleft palate, spina bifida and exencephalyincidence were 62.5%, 45.83% and 4.16% range in the group which received CP plus melatonin (10 mg/kg), respectively. In addition, theincidence of skeletal anomalies including limb, vertebral, and sternaldefects were decreased by melatonin and carvedilol. The mean weight and length of animal fetuses that had received melatonin and carvedilol were significantly greater than those receiving only CP. It is concluded; carvedilol has a significant effect in preventing CP-induced malformations and in cases like CP-induced exencephaly, cleft palate and spina bifidahas better prophylactic effect than melatonin, but this improvement is not significant.