Analysis of mutations and bone marrow micronuclei in Big Blue rats fed leucomalachite green.

Leucomalachite green (LMG) is the major metabolite of malachite green (MG), a triphenylmethane dye that has been used widely as an antifungal agent in the fish industry. Concern over MG and LMG is due to the potential for consumer exposure, suggestive evidence of tumor promotion in rodent liver, and suspicion of carcinogenicity based on structure-activity relationships. In order to evaluate the risks associated with exposure to LMG, female Big Blue rats were fed up to 543 ppm LMG; groups of these rats were killed after 4, 16, or 32 weeks of exposure and evaluated for genotoxicity. We previously reported that this treatment resulted in a dose-dependent induction of liver DNA adducts, and that the liver lacI mutant frequency (MF) was increased, but only in rats fed 543 ppm LMG for 16 weeks. In the present study, we report the results from lymphocyte Hprt mutant assays and bone marrow micronucleus assays performed on these same rats. In addition, we have determined the types of lacI mutations induced in the rats fed 543 ppm LMG for 16 weeks and the rats fed control diet. No significant increases in the frequency of micronuclei or Hprt mutants were observed for any of the doses or time points assayed. Molecular analysis of 80 liver lacI mutants from rats fed 543 ppm LMG for 16 weeks revealed that 21% (17/80) were clonal in origin and that most (55/63) of the independent mutations were base pair substitutions. The predominant type of mutation was G:C --> A:T transition (31/63) and the majority (68%) of these involved CpG sites. When corrected for clonality, the 16-week lacI mutation frequency (36 +/- 10) x 10(-6) in treated rats was not significantly different from the clonally corrected control frequency (17 +/- 9 x 10(-6); P = 0.06). Furthermore, the lacI mutational spectrum in treated rats was not significantly different from that found for control rats (P = 0.09). Taken together, these data indicate that the DNA adducts produced by LMG in female rats do not result in detectable levels of genotoxicity, and that the increase in lacI MF observed previously in the liver of treated rats may be due to the disproportionate expansion of spontaneous lacI mutations.

Mutagenicity and carcinogenicity in relation to DNA adduct formation in rats fed leucomalachite green.

Leucomalachite green is a persistent and prevalent metabolite of malachite green, a triphenylmethane dye that has been used widely as an antifungal agent in the fish industry. Concern over the use of malachite green is due to the potential for consumer exposure, evidence suggestive of tumor promotion in rodent liver, and suspicion of carcinogenicity based on structure-activity relationships. Our previous study indicated that feeding rodents malachite or leucomalachite green resulted in a dose-related increase in liver DNA adducts, and that, in general, exposure to leucomalachite green caused an increase in the number and severity of changes greater than was observed following exposure to malachite green. To characterize better the genotoxicity of leucomalachite green, female Big Blue rats were fed leucomalachite green at doses of 0, 9, 27, 91, 272, or 543 ppm for up to 32 weeks. The livers were analyzed for lacI mutations at 4, 16, and 32 weeks and DNA adducts at 4 weeks. Using a 32P-postlabeling assay, we observed a dose-related DNA adduct in the livers of rats fed 91, 272, and 543 ppm leucomalachite green. A approximately 3-fold increase in lacI mutant frequency was found in the livers of rats fed 543 ppm leucomalachite green for 16 weeks, but significant increases in mutant frequencies were not found for any of the other doses or time points assayed. We also conducted 2-year tumorigenesis bioassays in female and male F344 rats using 0, 91, 272, and 543 ppm leucomalachite green. Preliminary results indicate an increasing dose trend in lung adenomas in male rats treated with leucomalachite green, but no increase in the incidence of liver tumors in either sex of rat. These results suggest that the DNA adduct formed in the livers of rats fed leucomalachite green has little mutagenic or carcinogenic consequence.

Effect of betamethasone on maternal glucose.

OBJECTIVE: To determine the effect of single and multiple betamethasone courses on maternal fasting and postprandial glucose values. STUDY DESIGN: A prospective cohort study was performed in women receiving betamethasone at 24-34 weeks' gestation. Fasting and 1-h postprandial capillary glucose values were obtained daily following betamethasone therapy for hospitalized patients. A control group comprised outpatients who underwent weekly fasting and postprandial assessments for 3 weeks. Fasting and 1-h postprandial capillary glucose values were compared between control and betamethasone patients using an unpaired t test. RESULTS: Thirty-five women received a single course of therapy, 19 received multiple courses and 28 served as controls. Mean fasting glucose values for control patients fell within a narrow range of 81.6 +/- 10.3 to 82.2 +/- 6.4 mg/dl for weeks 1-3. Of women receiving betamethasone, 59% of fasting glucose values were greater than 90 mg/dl as compared to 16% of control fasting values (p < 0.00 1, chi2 test). Mean 1-h postprandial values for control women ranged from 107.7 +/- 15.1 to 112.3 +/- 20.0 mg/dl for weeks 1-3. Mean 1-h postprandial glucose values were < or = 140 mg/dl following one, two or three courses of betamethasone therapy. CONCLUSIONS: Betamethasone resulted in an acute increase in fasting glucose following a single course of betamethasone, whereas two or more courses of therapy resulted in a continuous elevation of fasting glucose values. One-hour postprandial values were not clinically abnormal.

Gene- and tissue-specificity of mutation in Big Blue rats treated with the hepatocarcinogen N-hydroxy-2-acetylaminofluorene.

In a previous study, we found that treating transgenic Big Blue rats with the hepatocarcinogen N-hydroxy-2-acetylaminofluorene (N-OH-AAF) produced the same major DNA adduct in the target liver and the nontarget spleen lymphocytes and bone marrow cells, induced lacI mutants in the liver, and induced much lower frequencies of lacI and hprt mutants in spleen lymphocytes. In the present study, sequence analysis was conducted on lacI DNA and hprt cDNA from the mutants, to determine the mutational specificity of N-OH-AAF in the rat. All the mutation spectra from N-OH-AAF-treated rats differed significantly from corresponding mutation profiles from untreated animals (P = 0.02 to P < 0.0001). Although there were similarities among the mutational patterns derived from N-OH-AAF-treated rats (e.g., G:C --> T:A transversion was the most common mutation in all mutation sets), there were significant differences in the patterns of basepair substitution and frameshift mutation between the liver and spleen lymphocyte lacI mutants (P = 0.02) and between the spleen lymphocyte lacI and hprt mutants (P = 0.04). Also, multiplex PCR analysis of genomic DNA from the hprt mutants indicated that 12% of mutants from treated rats had major deletions in the hprt gene; no corresponding incidence of large deletions was evident among lacI mutations. All the mutation profiles reflect the general mutational specificity of the major DNA adduct formed by N-OH-AAF. The differences between N-OH-AAF mutation in the endogenous gene and transgene can be partially explained by the structures of the two genes. The tissue-specificity of the mutation spectra may contribute to targeting tumor formation to the liver. Environ. Mol. Mutagen. 37:203-214, 2001. Published 2001 Wiley-Liss, Inc.

Urinary interleukin-8 with asymptomatic bacteriuria in pregnancy.

OBJECTIVE: To evaluate urinary interleukin-8 (IL-8), an inflammatory cytokine, as a screening method for detecting asymptomatic bacteriuria in pregnancy. METHODS: Clean-catch urine samples from 200 pregnant women undergoing screening for asymptomatic bacteriuria were evaluated by urine culture, urine dipstick analysis, and measurement of IL-8. Interleukin-8 levels were measured by a chemiluminescent immunoassay (Immulite IL-8, Diagnostic Products Corp., Los Angeles, CA), and a receiver operating characteristic curve was used to determine the optimal cutoff point. Asymptomatic bacteriuria was defined as at least 100,000 colony-forming units of a single organism per mL. Dipstick testing included nitrite assessment as positive or negative and leukocyte esterase as negative, trace, 1+, 2+, or 3+. Dipstick testing was considered positive if nitrite was positive or leukocyte esterase was trace or greater. Sensitivities, specificities, positive and negative predictive values were determined for urinary leukocyte esterase and nitrite and compared with those of IL-8. chi(2) and Mann-Whitney U tests were used for statistical analyses. RESULTS: Twenty women were identified with asymptomatic bacteriuria by urine culture. The median urinary IL-8 levels for women with and without asymptomatic bacteriuria were 356 pg/mL and 125 pg/mL, respectively (P <.01, Mann-Whitney U test). Using an optimal cutoff point of 264 pg/mL, IL-8 had a sensitivity, specificity, positive and negative predictive value of 70%, 67%, 19%, and 95% for predicting asymptomatic bacteriuria. Urine dipstick analysis with either a positive leukocyte esterase or nitrite had a sensitivity, specificity, positive and negative predictive value of 45%, 62%, 12%, and 91%, respectively, for detecting asymptomatic bacteriuria. The differences between these testing methods were not statistically significant. CONCLUSION: Urinary interleukin-8 is not an acceptable screening method for asymptomatic bacteriuria in pregnancy because it fails to detect 30% of women with this condition.

Repeated fetal betamethasone treatment and birth weight and head circumference.

OBJECTIVE: To assess the effect of repeated courses of betamethasone on birth weight and head circumference. METHODS: We conducted a historical cohort study of inpatients receiving betamethasone therapy over 5 years. We compared birth weights and head circumferences of infants whose mothers received one course of betamethasone with those of infants whose mothers received multiple courses. Multiple regression analysis was used to adjust for potential confounding variables. Sufficient power (80%) existed to detect a 20% difference between the groups (alpha = 0.05). RESULTS: Mean birth weights (+/-SD) were 1717 +/- 707 g in the single-course group (n = 107) and 1783 +/- 647 g in the multiple-course group (n = 45) (P =.59, Student t-test). Mean head circumference was 28.2 +/- 3.6 cm in the single-course group and 29.2 +/- 2.9 cm in the multiple-course group (P =.15, Student t-test). In regression analysis, birth weights (1757 g and 1752 g) and head circumferences (28.5 cm and 29.0 cm) did not differ significantly different between the single-course and multiple-course groups. CONCLUSION: Multiple courses of betamethasone do not reduce birth weight or head circumference in neonates compared with single-course therapy.

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.

DNA adduct formation and molecular analysis of in vivo lacI mutations in the mammary tissue of Big Blue rats treated with 7, 12-dimethylbenz[a]anthracene.

Recently we compared the lacI and Hprt mutant frequencies (MFs) and types of mutations in lymphocytes of Big Blue((R)) (BB) rats exposed to 7,12-dimethylbenz[a]anthracene (DMBA) under conditions that result in mammary gland tumors. In this study, we have examined the target mammary tissue for DMBA-induced DNA adducts, lacI MF and types of lacI mutations. Seven-week-old female BB rats were given single doses of 0, 20 or 130 mg/kg DMBA by gavage and the DNA adducts and lacI MFs in the mammary tissue were measured over a period of 14 days and 18 weeks, respectively, following treatment. The lacI MF in the mammary tissue increased for 10 weeks and then remained relatively constant; 130 mg/kg DMBA produced a 14-fold increase in the MF (255 +/- 50 x 10(-6) p.f.u.) over control MF (18. 3 +/- 4 x 10(-6) p.f.u.). (32)P-post-labeling analysis of DNA from mammary tissue and splenic lymphocytes of treated rats revealed two major adducts. Comparison of these adducts with DMBA standards indicated that the adducts formed by DMBA involved both G:C and A:T base pairs. DNA sequencing revealed that the majority of DMBA-induced lacI mutations were base pair substitutions and that A:T-->T:A (44% of the independent mutations) and G:C-->T:A (24% of the independent mutations) transversions were the predominant types. Furthermore, the mutational results revealed a 'hotspot' for a G-->T mutation in codon 95 (GTG-->TTG) of the lacI gene in mammary tissue. These results suggest that DMBA is highly mutagenic to lacI in mammary tissue and that adducts with both G:C and A:T base pairs participate in forming mutations in DMBA-treated BB rats.

Cardiac troponin T levels in umbilical cord blood.

OBJECTIVE: We sought to determine cardiac troponin T concentrations in umbilical cord plasma from normal and complicated pregnancies. STUDY DESIGN: At the time of delivery, umbilical cord arterial and venous samples were collected from 209 neonates, and cardiac troponin T levels were measured by immunoassay. Comparisons of clinical factors were made between neonates with normal and elevated cardiac troponin T levels. Significance was deemed present at P <.05. RESULTS: Twelve neonates had elevated cardiac troponin T levels. Exposure to magnesium sulfate was associated with an elevated cardiac troponin T level (relative risk, 33.2; 95% confidence interval, 7.7-143). CONCLUSIONS: Cardiac troponin T levels were elevated in neonates exposed to magnesium sulfate in utero. The explanation of this finding and its clinical significance are unknown. Characterization of fetal and neonatal troponin T requires further study.

Prenatal diagnosis of thrombocytopenia absent radius (TAR) syndrome and vaginal delivery.

A fetus with bilateral radial aplasia was identified on routine ultrasound. The diagnosis of thrombocytopenia absent radius (TAR) syndrome was confirmed with cordocentesis. The differential diagnosis of radial aplasia and prenatal tests available to assist with management are discussed. Cordocentesis offered useful information in the management of this case for both diagnosis and in deciding the route of delivery. We believe our case represents the first prenatal diagnosis of TAR syndrome in which vaginal delivery of a liveborn infant was intentionally allowed. Caesarean delivery may not be necessary for all fetuses diagnosed with TAR syndrome.

Comparison of mutant frequencies and types of mutations induced by thiotepa in the endogenous Hprt gene and transgenic lacI gene of Big Blue rats.

The utility of the lacI transgene of Big Blue rats as a reporter of in vivo mutation was evaluated by comparing the frequency and types of mutations induced by thiotepa in the transgene and the endogenous Hprt gene. Transgenic rats were given i.p. injections of 1.4 mg/kg of thiotepa three times per week over a period of 4 weeks (a total dose of 16.8 mg/kg); 1 week after the last injection, mutation assays were performed on spleen lymphocytes isolated from the animals. Thiotepa treatment increased the lacI mutant frequency from 34.8 +/- 4.1 x 10(-6) in control animals to 140.9 +/- 64.8 x 10(-6) (p = 0.0020) and the Hprt mutant frequency from 3.5 +/- 1.5 x 10(-6) to 41.1 +/- 23.2 x 10(-6) (p = 0.0028). Sequence analysis of lacI mutant DNA and Hprt mutant cDNA produced similar overall mutation patterns: G:C-->T:A transversion was the most common base pair substitution (32% of independent mutations in the lacI gene and 28% of Hprt mutations), and deletions and insertions accounted for 34% of mutations in the lacI gene and 28% in the Hprt gene. The majority of thiotepa-induced base pair substitutions in the Hprt gene occurred with the mutated purine on the non-transcribed DNA strand, while no strand-related bias was found for mutations in the lacI gene. Substitutions at G:C base pairs in the lacI gene, but not in the Hprt gene, were found disproportionately in CpG sites. In addition, multiplex polymerase chain reaction analysis of genomic DNA from the Hprt mutants indicated that 34% had relatively large deletions; none of these deletions was detected by the cDNA analysis. The results indicate that the frequency of thiotepa-induced mutants in Big Blue rats was 2.8-fold greater in the lacI gene than in the Hprt gene. Although the Hprt gene recovered a fraction of large deletions not found among the lacI mutants, the effects of transcription-coupled DNA repair in the Hprt gene and the targeting of base pair substitutions to G:C base pairs in CpG sites may have contributed to the higher mutant frequencies induced by thiotepa in the lacI transgene compared with the Hprt gene.

Comparison of in vivo mutagenesis in the endogenous Hprt gene and the lacI transgene of Big Blue(R) rats treated with 7, 12-dimethylbenz[a]anthracene.

The lacI transgene of Big Blue(R) (BB) rats was evaluated as a reporter of in vivo mutation by comparing mutant frequencies (MFs) in it and in the endogenous Hprt gene. Seven-week old female BB rats were given single doses of 0, 20, 75 and 130 mg/kg of 7, 12-dimethylbenz(a)anthracene (DMBA) by gavage, and Hprt and lacI MFs in splenic lymphocytes were measured over a period of 18 weeks. The Hprt MFs in treated rats increased for 10 weeks and then declined; 130 mg/kg of DMBA produced a maximum Hprt MF of 168+/-11.4x10-6 clonable lymphocytes, while the MF in control rats was 7.4+/-1. 5x10-6. DMBA exposure of generic F344 rats resulted in a similar time-course of mutant induction but produced about 50% higher Hprt MFs with the 75 and 130 mg/kg doses. In contrast, the lacI MFs increased for 6 weeks and then remained relatively constant; 130 mg/kg of DMBA produced a maximum increase in lacI MF of 341+/-83x10-6 PFU compared with 25+/-5x10-6 PFU in control rats. The Hprt mutant frequencies in DMBA-treated BB and F344 rats were significantly increased over control values for every dose-time combination examined, while only the 130 mg/kg dose consistently produced lacI MFs that were significantly above the controls. In addition, the fold-increase in MF for treated vs. control rats was two times higher for the Hprt gene than the lacI gene due to the higher MFs in the lacI gene of control rats. Differences between the lacI and Hprt genes in the kinetics of mutant induction, in the frequency of induced mutants, and in the sensitivity of mutant detection could be explained at least partially by the properties of these two genes.

Comparison of the types of mutations induced by 7,12-dimethylbenz[a]anthracene in the lacI and hprt genes of Big Blue rats.

An important question regarding the use of transgenic reporter genes to detect mutation in rodents is how the types of mutations recovered in transgenes compare with the types of mutations found in endogenous genes. In this study, we examined mutations induced by 7,12-dimethylbenz[a]anthracene in the lacI transgene and the endogenous hprt gene of lymphocytes from Big Blue rats and in the hprt gene of lymphocytes from nontransgenic Fischer 344 rats. The overall mutation profiles found in these genes were remarkably similar: the majority of mutations were base pair substitutions, with the most common mutation being A:T-->T:A transversion. Differences were found for the mutational profiles in the endogenous gene and transgene with respect to the location of the mutations and the orientation of basepair substitutions in the DNA strands. In most cases, these differences could be explained by the nature of the target genes. The results support the use of the lacI transgene for detecting in vivo mutation.

Molecular analysis of lacI mutations in Rat2 cells exposed to 7,12-dimethylbenz[a]anthracene: evidence for DNA sequence and DNA strand biases for mutation.

The Rat2 cell line carries 50-70 stably integrated copies per cell of a lambda/lacI shuttle vector as a target for mutagenicity testing. Rat2 cells were exposed to 1 and 10 micrograms/ml of 7,12-dimethylbenz[a]anthracene (DMBA) for 24 h at 37 degrees C in the presence of primary rat hepatocytes, and grown to confluence. The shuttle vector was rescued from untreated and mutagen-treated cells and mutant frequencies were determined. The low and high doses of DMBA induced mutant frequencies that were 7-fold (25 +/- 4.9 x 10(-5)) and 33-fold (127 +/- 19.9 x 10(-5)) higher, respectively, than the spontaneous mutant frequency (3.8 +/- 0.7 x 10(-5)). DNA sequence analysis of the DMBA-induced lacI- mutants indicated that they contained mainly basepair substitution mutations at A:T and G:C, and that A:T-->T:A and G:C-->T:A transversions were the predominant types. In addition, 23 of 28 (82%) A:T basepair substitution mutations occurred with the mutated dA, the putatively adducted base, on the coding strand. Furthermore, 20 of the 28 (71%) A:T mutations had the mutated dA flanked 5' by a dC, and 17 of these were A:T-->T:A transversions, suggesting a sequence preference for this mutation. Except for a higher proportion of G:C-->A:T transitions in the low dose data, the mutational profiles from low and high doses of DMBA were similar. These results indicate that DMBA mutagenesis in the lacI gene of Rat2 cells displays distinct DNA sequence and DNA strand preferences.

A mutation in the p53 tumor suppressor gene of AHH-1 tk+/- human lymphoblastoid cells.

Loss-of-function mutations in the p53 tumor suppressor gene result in an altered response to DNA-damaging agents. Included in the mutant p53 phenotype are the loss of the G1 checkpoint and delayed apoptotic cell death, characteristics we have consistently observed in the AHH-1 tk+/- cell line following exposure to DNA-damaging agents. In order to determine the functional status of p53 in the AHH-1 tk+/- cell line, molecular analysis (single-strand conformational polymorphism [SSCP] and sequence analysis) was performed on exons 5-9 of the p53 gene. In addition, the status of the p53 gene in the closely related lymphoblast line, MCL-5, which, in our hands, has a much higher spontaneous rate of apoptosis than AHH-1 tk+/-, was also determined by molecular analysis. Initial SSCP analysis of AHH-1 tk+/- revealed an abnormal migration pattern of exon 8 when compared to a wild-type control. Subsequent sequence analysis indicated that a base-pair substitution (CGG-->TGG) mutation had occurred at codon 282, a reported "hot spot' for 5-methylcytosine mutations in the human p53 gene. Neither SSCP nor sequence analysis of exons 5-9 of MCL-5 indicated any differences from wild-type DNA. These results suggest that the lack of a G1 arrest and the delayed entrance into apoptosis observed in chemically-exposed AHH-1 tk+/- cells are, at least partially, accounted for by a loss-of-function mutation in the p53 gene.