Reaction of the antitumor antibiotic CC-1065 with DNA: structure of a DNA adduct with DNA sequence specificity.

Sequence-dependent variations in DNA revealed by x-ray crystallographic studies have suggested that certain DNA-reactive drugs may react preferentially with defined sequences in DNA. Drugs that wind around the helix and reside within one of the grooves of DNA have perhaps the greatest chance of recognizing sequence-dependent features of DNA. The antitumor antibiotic CC-1065 covalently binds through N-3 of adenine and resides within the minor groove of DNA. This drug overlaps with five base pairs for which a high sequence specificity exists.

Inherent tumorigenic and metastatic properties of rat-1 and rat-2 cells.

Rat-1 and Rat-2 cells have been used in many studies of in vitro transformation and are widely assumed to be nontumorigenic because of their low incidence of focus formation, their poor growth in soft agar, and their reported failure to form tumors in animals. We examined more carefully the relationship between the in vitro and in vivo behavior of these cells and found that in spite of their in vitro characteristics, injection of these cells into Fischer rats invariably produced invasive tumors which frequently metastasized. When cells from primary tumors or metastases were cultured in vitro, the resultant cell lines were usually morphologically indistinguishable from parental cells and neither formed foci nor grew in soft agar. Thus, in vitro growth patterns do not correlate well with in vivo behavior in these cells and their inherent tumorigenicity warrants caution in the interpretation of results of in vitro transformation studies.

Regulation of a metallothionein-rasT24 fusion gene by zinc results in graded alterations in cell morphology and growth.

We constructed fusion genes consisting of the mouse metallothionein I (MT) 5' region and the coding region of either the human H-ras gene (c-rasP3) or a mutated allele (c-rasT24); both ras genes lacked the initial (non-coding) exon and the first 30 bp of the non-coding region of the second exon. Transfection of Rat-1 cells produced foci only with pMT-rasT24, and selection in soft agar yielded clones in which MT-rasT24 expression was zinc-regulatable. In response to increasing concentrations of ZnSO4, these lines showed increasingly altered morphology (conversion to fusiform or spheroidal morphology), progressively higher maximal cell density, and an increasingly greater fraction of cells in the S + G2 + M portion of the cell cycle at high density. MT-rasT24 RNA levels in zinc-responsive lines were increased between 4- and 6-fold by the addition of ZnSO4 (final concentration = 100 microM) to the medium. Replating cells in the absence of zinc reversed the biological effects and resulted in reduction in MT-rasT24 RNA levels. Thus, graded alterations in phenotype result from increasing levels of MT-rasT24 gene expression.

Genetic toxicity evaluation of octamethylcyclotetrasiloxane.

Octamethylcyclotetrasiloxane (OMCTS; CAS No. 556-67-2) was evaluated in a genetic toxicity battery. In preincubation tests with Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538, no mutagenicity was detected (maximum dose = 5 mg/plate) with or without S9 in two independent trials. Treatment of cultured Chinese hamster ovary (CHO) cells was limited by cytotoxicity at OMCTS concentrations greater than 0.003 mg/mL without S9 and 0.03 mg/mL with S9. CHO cells treated with up to 0.003 mg/mL without S9 and 0.03 mg/mL with S9 showed no significant dose-related increases in chromosomal aberration frequencies. No significant dose-related increases in sister chromatid exchanges (SCEs) occurred in OMCTS-treated CHO cells (maximum OMCTS concentration = 0.003 mg/mL without S9; 0.03 mg/mL with S9). Therefore, OMCTS was concluded to be negative in the SCE assay. In a screen for in vivo clastogenic potential, Sprague-Dawley rats received 700 ppm OMCTS by whole-body vapor inhalation 6 hr daily for 5 days. A negative control group received filtered air on the same schedule. A positive control group was exposed to filtered air on the same schedule and received cyclophosphamide 24 hr before termination. The OMCTS-treated animals were terminated 6 and 24 hr after the final exposure. Positive and negative control animals were terminated 24 hr after the last exposure. No significant, treatment-related increases in chromosomal aberrations were detected. The results of these studies indicate that OMCTS does not possess significant in vitro genotoxic potential. No adverse genetic findings were seen in the in vivo screen for chromosome aberrations.

Comparison of properties of the in vitro and cellular anthramycin-DNA adducts and characterization of the reaction of anthramycin with chromatin.

The reaction of anthramycin with DNA has been examined to determine the chemical identity of the adduct which forms in a living cell and to observe the effects of the nucleosome structure of chromatin on drug binding. The chemical identity of the cellular adduct was probed by comparing various properties of the cellular adduct to properties of the known, in vitro adduct. The effect of the histones on anthramycin binding was investigated by time-course binding reactions. Results indicate that the properties of the cellular anthramycin-DNA adduct are similar to the in vitro adduct. The histone proteins associated with DNA in chromatin were found to decrease both the reaction kinetics and the final levels of anthramycin binding. Anthramycin reacts appreciably with nucleosome core DNA, but appears to exhibit a preference for linker DNA.

Interpreting the toxicologic significance of alterations in anogenital distance: potential for confounding effects of progeny body weights.

Anogenital distance (AGD) is an endpoint that was recently added to the U.S. EPA testing guidelines for reproductive toxicity studies. This endpoint is sensitive to hormonal effects of test chemicals. It is possible that apparent alterations in AGD might occur after treatment with agents that affect overall pup body size. In such cases, hormonal activity might be associated incorrectly with the test treatment. The analyses in this report evaluated statistical correlations between pup body weight and AGD in control litters. AGDs were measured on postnatal day 1 in 1501 pups derived from 113 untreated female Sprague-Dawley rats in two independent two-generation reproductive toxicity studies. Significant correlations were detected between AGD and body weight and between AGD and the cube root of body weight. In males, AGD increased 0.26 mm for each 1 g increase in body weight. In females, AGD increased 0.13 mm per 1 g increase in body weight. Although there were essentially no differences between the regression models developed to predict AGD in either males or females using body weight as a covariate and those based on the cube root of body weight, such similarities in predictivity might not occur in larger animals with broader weight ranges than those encountered in this analysis. Normalization of AGD by dividing by body weight significantly overcompensated for differences in body size. Normalizing with the cube root of body weight resulted in an AGD/cube root of body weight ratio that was constant across the range of body weights observed in this study. In conclusion, as a preferred method to account for body size effects on AGD, analysis of covariance is recommended. If a normalization is done directly, the ratio of AGD to the cube root of body weight is the more appropriate metric.

Evaluation of carbendazim for gene mutations in the Salmonella/Ames plate-incorporation assay: the role of aminophenazine impurities.

Benomyl (methyl [1-[(butylamino)carbonyl]-1H-benzimidazol-2- yl]carbamate) and its major metabolite carbendazim (methyl 2-benzimidazolecarbamate) are major agricultural systemic fungicides. These compounds inhibit fungal microtubular function and thereby cause nondisjunction of chromosomes at cell division. Several investigators have proposed that these compounds can also cause gene mutations (base-pair substitutions). In this laboratory, no mutagenic activity was observed with either benomyl (analytical grade) or Benlate (samples tested up to 500 and 1200 micrograms/plate, respectively, the limit of cytotoxicity) in the Salmonella/Ames plate-incorporation test in either base-pair substitution (TA100 and TA1535) or frameshift-sensitive (TA98 and TA1537) strains with or without S9 metabolic activation. However, some carbendazim preparations caused mutations in frameshift-sensitive strains at very high concentrations (> or = 5000 micrograms/plate) with metabolic activation. The mutagenic activity was not due to the major carbendazim metabolite, methyl (5-hydroxy-1H-benzimidazol-2-yl)carbamate (5-OH MBC), since 5-OH MBC was not mutagenic with (up to 20,000 micrograms/plate) or without (up to 16,000 micrograms/plate) activation. Subsequently, two highly mutagenic contaminants, 2,3-diaminophenazine (DAP) and 2-amino-3-hydroxyphenazine (AHP) were detected in mutagenic carbendazim samples. In those samples, DAP and AHP contaminant levels ranged as high as 46.5 and 11.6 ppm, respectively. No evidence of mutagenicity could be detected in preparations in which the DAP content was < 1.8 ppm. The mutagenic activity of these two contaminants was further investigated in strain TA98. Without activation, DAP and AHP were positive at test concentrations as low as 5 and 10 micrograms/plate, respectively. In the presence of S9, mutations were detected at much lower concentrations (beginning at 0.025 and 0.05 microgram/plate, respectively). These results indicate that carbendazim samples containing DAP or AHP at levels as low as 5 or 10 ppm, respectively, would be positive in the Salmonella/Ames test with activation when tested at 5000 micrograms/plate. Purified carbendazim is not mutagenic.

Evaluation of benomyl and carbendazim in the in vivo aneuploidy/micronucleus assay in BDF1 mouse bone marrow.

Benomyl and its active metabolite carbendazim were investigated in BDF1 mouse bone marrow to establish whether micronuclei induced by these fungicides are caused by clastogenic or aneugenic events. Micronuclei were evaluated for kinetochores using immunofluorescent antikinetochore antibodies. Kinetochore positive (K+) micronuclei are likely to arise from chromosome loss since they presumably contain intact kinetochores and are indicative of aneuploidy. Conversely, kinetochore negative (K-) micronuclei are mostly likely to contain acentric chromosome fragments arising primarily from clastogenic damage. Benomyl and carbendazim were administered as single oral doses of 0.3, 8.6 or 17.2 mmol/kg (for benomyl, equivalent to 100, 2500 or 5000 mg/kg; for carbendazim, equivalent to 66, 1646 or 3293 mg/kg). Both compounds were positive in the micronucleus test at doses of 8.6 and 17.2 mmol/kg, and an average of 82% (benomyl) and 87% (carbendazim) of the total micronucleated polychromatic erythrocytes were K+. No effects were seen with either fungicide at 0.3 mmol/kg. These results are analogous to findings with known aneugens such as vincristine but are in contrast to results with classical clastogens such as cyclophosphamide. Thus, benomyl and carbendazim induce micronuclei in mouse bone marrow cells primarily through an aneugenic mechanism.

DNA sequence specificity of the pyrrolo[1,4]benzodiazepine antitumor antibiotics. Methidiumpropyl-EDTA-iron(II) footprinting analysis of DNA binding sites for anthramycin and related drugs.

Anthramycin, tomaymycin, and sibiromycin are members of the pyrrolo[1,4]benzodiazepine [P(1,4)B] antitumor antibiotic group. These drugs bind covalently through N2 of guanine and lie within the minor groove of DNA [Petrusek, R. L., Anderson, G. L., Garner, T. F., Fannin, Q. L., Kaplan, D. J., Zimmer, S. G., & Hurley, L. H. (1981) Biochemistry 20, 1111-1119]. The DNA sequence specificity of the P(1,4)B antibiotics has been determined by a footprinting method using methidiumpropyl-EDTA-iron(II) [MPE.Fe(II)], and the results show that each of the drugs has a two to three base pair sequence specificity that includes the covalently modified guanine residue. While 5'PuGPu is the most preferred binding sequence for the P(1,4)Bs, 5'PyGPy is the least preferred sequence. Footprinting analysis by MPE.Fe(II) reveals a minimum of a three to four base pair footprint size for each of the drugs on DNA with a larger than expected offset (two to three base pairs) on opposite strands to that observed in previous analyses of noncovalently bound small molecules. There is an extremely large enhancement of MPE.Fe(II) cleavage between drug binding sites in AT rich regions, probably indicating a drug-induced change in the conformational features of DNA which encourages interaction with MPE.Fe(II). In the presence of sibiromycin or tomaymycin the normally guanine-specific methylene blue reaction used in Maxam and Gilbert sequencing cleaves at other bases in defined positions relative to the drug binding sites. Finally, modeling studies are used to rationalize the differences and similarities in sequence specificities between the various drugs in the P(1,4)B group and their reactions with DNA.

Reaction of the antitumor antibiotic CC-1065 with DNA. Location of the site of thermally induced strand breakage and analysis of DNA sequence specificity.

CC-1065 is a unique antitumor antibiotic produced by Streptomyces zelensis. The potent cytotoxic effects of this drug are thought to be due to its ability to form a covalent adduct with DNA through N3 of adenine. Thermal treatment of CC-1065-DNA adducts leads to DNA strand breakage. We have shown that the CC-1065 structural modification of DNA that leads to DNA strand breakage is related to the primary alkylation site on DNA. The thermally induced DNA strand breakage occurs between the deoxyribose at the adenine covalent binding site and the phosphate on the 3' side. No residual modification of DNA is detected on the opposite strand around the CC-1065 lesion. Using the early promoter element of SV40 DNA as a target, we have examined the DNA sequence specificity of CC-1065. A consensus sequence analysis of CC-1065 binding sites on DNA reveals two distinct classes of sequences for which CC-1065 is highly specific, i.e., 5'PuNTTA and 5'AAAAA. The orientation of the DNA sequence specificity relative to the covalent binding site provides a basis for predicting the polarity of drug binding in the minor groove. Stereo drawings of the CC-1065-DNA adduct are proposed that are predictive of features of the CC-1065-DNA adduct elucidated in this investigation.

Characterization of an adduct between CC-1065 and a defined oligodeoxynucleotide duplex.

CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis. The drug binds covalently through N-3 of adenine and lies within the minor groove of DNA. Previous studies indicated that CC-1065 reacted with adenine in DNA to yield a thermally labile product that could be used to reveal its sequence specificity. These studies also provided insight into a DNA sequence (5'-CGGAGTTAGGGGCG-3') which should bind one molecule of CC-1065 in an unambiguous manner. This sequence, which contains the CC-1065 adenine binding site within the sequence 5'-TTA-3' was chemically synthesized together with the complementary strand. CC-1065 reacted with the oligoduplex to give an adduct that maintained the B-DNA form and had a final CD spectrum similar to those of the CC-1065 complexes formed with calf thymus DNA. The above 14mer was 5' end-labelled with 32P, annealed with its complementary strand, reacted with CC-1065 and heated. Drug-mediated strand breakage was evaluated on a sequencing gel. A single break occurred in the labelled strands to give a fragment that migrated as an 8.5mer; subsequent piperidine treatment produced a fragment that migrated as a 7mer, which is the size expected from the known binding of CC-1065 at adenine in 5'-TTA-3' sequences.