A comparative study of in vivo mutation assays: analysis of hprt, lacI, cII/cI and as mutational targets for N-nitroso-N-methylurea and benzo[a]pyrene in Big Blue mice.

We have compared the response of the native hprt gene and the lacI, cII, and cI transgenes in Big Blue B6C3F1 mice following treatment with either N-nitroso-N-methylurea (MNU) or benzo[a]pyrene (BaP). Three weeks after mutagen treatment splenic T cells were isolated from the animals, and samples were either cultured to measure mutation at the native hprt locus or used to extract genomic DNA for transgene mutation analysis. Phage rescued from extracted DNA were plated in the presence of 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside (X-gal) to score lacI mutations, or plated on a hflAB lawn to score cII and cI mutants. With MNU hprt mutant frequency increased in a dose-related, sublinear manner up to 78-fold above background at the highest dose tested (20 mg/kg). In comparison, the lacI transgene yielded only a 3.1-fold increase at this dose, and the cII and cI transgenes did not show any increase. With 150 mg/kg BaP a 5.8- and 8.7-fold increase in mutant frequency was observed at hprt and lacI, respectively, while only a 1.3-fold increase was observed at cII. DNA sequencing revealed an increase in GC-->TA transversions among the cII mutants, suggesting that the increase was related to BaP exposure. No significant increase in cI mutant frequency was observed. Therefore, the order of mutation assay sensitivity was hprt>lacI>cII/cI with MNU, and hprt approximately lacI> cII/cI with BaP. While the hflAB selection system offers significant advantages with respect to cost and effort when compared to the lacI assay, additional evaluation of its sensitivity is warranted.

Synthesis of a lacI gene analogue with reduced CpG content.

A lacI gene analogue with reduced CpG content has been synthesized. Codon usage in the lacI gene was manipulated to remove most CpG sites (82/95; 86%) while maintaining wild-type amino acid sequence. The double-stranded gene sequence was synthesized using standard beta-cyanoethyl phosphoramidite chemistry and subsequently cloned into pBR322. Bacterial promoter sequences with different levels of activity were attached upstream of the modified coding region to study its expression in E. coli. Production of lacI protein was confirmed in a lacI- E. coli strain by Western blot analysis and by measuring repression of the lacZ gene with the chromogenic lacZ indicator, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal). The modified lacI gene construct can be used as a genetic target in cultured mammalian cells or in transgenic animals to avoid high levels of background mutation associated with methylated CpG sequences. The construction scheme described here provides a general approach to remove CpG sequences from gene constructs when methylation is undesirable.

Mutagenic response of the endogenous hprt gene and lacI transgene in benzo[a]pyrene-treated Big Blue B6C3F1 mice.

Big Blue (BB) and generic B6C3F1 mice were given one to three i.p. injections of 50 mg/kg benzo[a]pyrene (B[a]P) in DMSO every other day to achieve cumulative doses of 50 to 150 mg/kg. Three weeks after treatment, the mutation frequency at the endogenous hprt gene and lacI transgene was measured in splenic T cells. Generic mice given 50, 100, and 150 mg/kg B[a]P displayed induced hprt frequencies (observed hprt frequency minus control frequency) of 5.5 +/- 1.0, 11 +/- 2.0, and 19 +/- 2.6 x 10(-6), respectively (average +/- SEM). In contrast, BB mice given 50 and 150 mg/kg B[a]P displayed induced hprt frequencies of 0.9 +/- 0.6 and 9.1 +/- 1.5 x 10(-6). 32P postlabelling revealed that the lower hprt response in BB mice correlated with lower amounts of BP-DNA adducts in spleen, liver, and lung 24 hours after B[a]P exposure. Western blot analysis of liver samples from B[a]P-treated mice suggests that the reduced adduct load in turn may be due to lower P450 1A1 levels in BB mice. The frequency of induced, nonsectored blue plaques (observed blue plaque frequency minus control frequency) in BB mice receiving 50 and 150 mg/kg B[a]P was 41 +/- 9 and 134 +/- 10 x 10(-6) (15- to 40-fold higher than the induced hprt frequency in the same treated animals). Sectored plaques were observed in both control and B[a]P groups but their frequency showed no relationship to dose (sectored frequency in all groups was approximately 20 x 10(-6)). To test whether persistent DNA adducts in the packaged lambda vector were contributing to the observed blue plaque frequency, purified lambda-LIZ DNA was treated in vitro with B[a]P diol epoxide (BPDE), packaged, and plated on E. coli lawn cells. Treatment with BPDE did not produce significant increases in homogeneous blue plaques, suggesting that the majority of mutants obtained from B[a]P-treated BB mice occurred in vivo. These results indicate that B[a]P exposure produces many more mutations at the lacI transgene than at the endogenous hprt locus.

Relative sensitivity of the endogenous hprt gene and lacI transgene in ENU-treated Big Blue B6C3F1 mice.

Three-week-old Big Blue (BB) B6C3F1 mice were given a single i.p. injection of ENU. Three weeks later, splenic T cells were isolated from each animal by ficoll gradient centrifugation and divided into two samples. One sample was cultured to measure hprt- mutation and the other was used to extract DNA for lacI- analysis. T cells from BB mice exposed to 0, 4.5, 13.5, and 40 mg ENU/kg (9 or 10 animals per group) displayed dose-related increases in the frequency of both hprt- and lacI- mutations. Within each treatment group, the ENU-induced mutation frequency (average observed mutation frequency minus average control frequency) was remarkably similar at the two loci. This suggests that treatments that increase mutation frequency at the endogenous hprt gene also produce similar incremental increases at the BB lacI transgene. However, because of the ten-fold higher spontaneous mutation rate at lacI, the fold-increase over background produced by ENU at this locus was significantly less than the fold-increase produced at hprt. For example, the 4.5 mg ENU/kg treatment produced a 5.2-fold increase above background at hprt (P = 0.001), whereas only a 1.5-fold increase was produced at lacI (P = 0.140). Consequently, mutagenic insults that produce up to a fivefold increase in mutation frequency at an endogenous locus may be difficult to detect at the lacI transgene. Finally, the ENU-induced response at hprt in BB mice was identical to that in generic B6C3F1 mice, suggesting that there are no inherent differences between transgenic and normal mice in their response to this mutagenic agent.

Proliferating cell nuclear antigen in developing and adult rat cardiac muscle cells.

During early development, rat cardiac muscle cells actively proliferate. Shortly after birth, division of cardiac muscle cells ceases, whereas DNA synthesis continues for approximately 2 weeks at a progressively diminishing rate. Little DNA synthesis or cell division occurs in adult cardiocytes. Thus, developing cardiac muscle cells are an ideal system in which to examine the expression of cell cycle-regulated genes during development. We chose to examine proliferating cell nuclear antigen (PCNA), a gene expressed at the G1/S phase boundary of the cell cycle. Northern blots of RNA from cardiac muscle cells from 18-day-old rat fetuses and from day 0, 5, and 14 neonatal as well as adult rat hearts revealed that the PCNA mRNA was found in cardiac muscle cells from all ages. However, because it was possible that this was a result of fibroblast PCNA gene expression, we used reverse transcription followed by polymerase chain reaction to see if it was possible to detect the message for PCNA in cardiac muscle cells from all ages. Because of the great sensitivity of this technique, RNA was recovered from 25 isolated adult cardiac muscle cells. Polymerase chain reaction amplification products for PCNA produced from the RNA isolated from these cells conclusively demonstrated that mRNA for this gene, which normally is associated with proliferating cells, is expressed in adult cardiac muscle cells that no longer divide. Furthermore, Western blot analysis demonstrated that the PCNA protein was found only in embryonic and neonatal cells and not in adult rat cardiac muscle cells. Therefore, it might be inferred from these data that PCNA might be regulated at the posttranscriptional level in adult cardiac muscle cells.

Norepinephrine-induced cardiac hypertrophy of the cat heart.

Norepinephrine administration causes progressive hypertrophy of the mammalian heart as measured by myocardial mass. The purpose of this study was to determine the growth response of the myocardial tissue components as well as the myocardial cell itself to norepinephrine. Young, adult cats were given low doses of norepinephrine in dextrose or dextrose alone twice daily for 15 days. On day 16, there were no changes in the animals body weight, right ventricular systolic pressure, right ventricular end-diastolic pressure, heart rate, cardiac index, or blood pressure. However, the right ventricle/body weight, the left ventricle/body weight and the total heart weight/body weight were increased significantly in the norepinephrine treated animals. The increase was on the order of 40%. The cardiac muscle cell was also significantly increased in size and both the right and left ventricular cardiac muscle cells exhibited a dramatic increase in size as measured by cross sectional area. Upon stereological examination it was found that the amount of hypertrophy as seen in the cardiac muscle cells was paralleled by the hypertrophy seen in the other tissue components of the myocardium. The volume density of the muscle cells, the interstitial components, as well as the blood vessel compartment were identical in the control and in the norepinephrine-treated groups. In conclusion, this study demonstrates that the response of the myocardium to norepinephrine is similar to that seen in response to a volume overload rather than that seen in response to pressure overload.

Observations on the development of the human atrioventricular node and bundle.

This light microscopic study of the cardiac junctional tissues was based on 27 human embryos, fetuses and postnatal hearts. Evidence was presented that superficial and deep portions of the postnatal AV node were derived from two cellular primordia in the posterior wall of the common atrium at the 6-mm stage. The small right primordia was associated with the right venous valve and give rise to the loosely organized superficial AV node that extended posteriorly to the coronary sinus ostium. A larger left primordia formed the more compact deep subdivision of the AV node located against the anulus fibrosus. In most postnatal hearts the two subdivisions are partially or completely fused to form the adult AV node. Failure of the nodal primordia to fuse during cardiogenesis may result in two separate nodal cell aggregates above the anulus. The present observations provide a rational explanation for the two AV nodal masses described in the literature and an additional specimen that is illustrated in this communication. An AV bundle was first identified in a 13-mm embryo and appeared to be derived from large clear cells of the posterior AV canal. At 25 mm the bundle formed a broad band across the top of the IV septum and continued into both ventricles. At this stage multiple cell strands penetrated the endocardial cushion to connect the AV bundle to the two nodal primordia. Failure of normal fusion between the AV node primordia and AV bundle can result in a variety of junctional anomalies including congenital heart block.