Profiling mitochondrial proteins in radiation-induced genome-unstable cell lines with persistent oxidative stress by mass spectrometry.

Previous work by Morgan and coworkers on radiation-induced genome instability in Chinese hamster ovary (CHO) cell lines showed that unstable LS-12 cells had persistently elevated levels of reactive oxygen species (ROS) that were likely due to dysfunctional mitochondria. To further investigate the correlation between radiation-induced genome instability and dysfunctional mitochondria, we performed quantitative high-throughput mass spectrometry on samples enriched in mitochondrial proteins from three chromosomally unstable CHO cell lines and their stable unirradiated GM10115 parental cell line. Out of several hundred identified proteins, sufficient data were collected on 74 mitochondrial proteins to test for statistically significant differences in their abundance between unstable and stable cell lines. The LS-12 cell line, which exhibited the highest level of ROS among the three unstable cell lines, was characterized by eight significantly down-regulated mitochondrial proteins, all associated with the TCA (tricarboxylic acid). Elevated levels of ROS relative to the unirradiated parental control were also statistically significant for the CS-9 cell line. The protein profile of CS-9 revealed five significantly up-regulated mitochondrial proteins, three of which are involved in oxidative phosphorylation. Elevation of ROS in the unstable 115 cell line was nearly as large as that seen in CS-9 cells but was not statistically significant. The mitochondrial protein profile of 115 cells showed significant down-regulation of acetyl-CoA-acetyltransferase, which was also down-regulated in LS-12, and two other proteins with abundances that were significantly different from control levels but were not directly related to either the TCA or oxidative phosphorylation. These results provide further evidence that elevated ROS and mitochondrial dysfunction are associated with radiation-induced genome instability; however, additional work is required to establish a firm mechanistic relationship between these end points.

Characterization of medium conditioned by irradiated cells using proteome-wide, high-throughput mass spectrometry.

Shedding, the release of cell surface proteins by regulated proteolysis, is a general cellular response to injury and is responsible for generating numerous bioactive molecules including growth factors and cytokines. The purpose of our work is to determine whether low doses of low-linear energy transfer (LET) radiation induce shedding of bioactive molecules. Using a mass spectrometry-based global proteomics method, we tested this hypothesis by analyzing for shed proteins in medium from irradiated human mammary epithelial cells (HMEC). Several hundred proteins were identified, including transforming growth factor beta (TGFB); however, no changes in protein abundances attributable to radiation exposure, based on immunoblotting methods, were observed. These results demonstrate that our proteomic-based approach has the sensitivity to identify the kinds of proteins believed to be released after low-dose radiation exposure but that improvements in mass spectrometry-based protein quantification will be required to detect the small changes in abundance associated with this type of insult.

Promotion of preneoplastic changes in liver by coal-derived organic mixtures applied to skin.

The promotion of preneoplastic hepatocyte foci was observed in rats neonatally initiated by a single intraperitoneal injection of benzo[a]pyrene (BP) or diethylnitrosamine (DEN) and exposed, from weaning, to repeated topical applications of coal-derived complex organic mixtures that are carcinogenic for mouse skin. Topical application of these mixtures in the absence of prior initiation did not cause significant induction of hepatocyte foci. These observations indicate the advantage of the neonatal rat hepatocarcinogenesis system for detecting promoting activity in carcinogenic mixtures and identify the existence of systemic tumorigenic risk from cutaneous contact with promoting agents.

Phenotypically selective promotion of diethylnitrosamine-initiated altered hepatocyte foci by dietary phenobarbital or a topically applied coal-derived organic mixture in male and female rats.

Relative frequencies of diethylnitrosamine (DEN)-initiated foci of altered hepatocytes appearing in response to promotion by either dietary phenobarbital or a topically applied coal-derived organic mixture (CDM) were investigated in male and female rats. The focus population was examined for two histochemical markers, elevated gamma-glutamyl transpeptidase [GG(+)] and iron exclusion [FE(-)], giving rise to 3 detectable focus phenotypes, i.e., GG(+) foci, FE(-) foci, and GG(+)/(FE(-) foci. Frequencies of the 3 phenotypes were quantitated through the use of serial frozen sectioning and computer-assisted image analysis. In agreement with our prior observations, cutaneous exposure to CDM or dietary phenobarbital promoted the expression of DEN-initiated foci. However, the current data showed that this promoting effect of CDM occurred only in females and was restricted to foci with the GG(+)/FE(-) phenotype. Dietary phenobarbital, on the other hand, promoted both the GG(+) and GG(+)/FE(-) phenotypes and was effective in both males and females, although a sex-related differential in the promoting efficiency of phenobarbital was also observed. The pronounced heterogeneity in the responses of the 3 focus phenotypes suggests that each phenotype is the consequence of a specific type of genomic alteration with a specific capacity to undergo phenotypic expression in response to a given promoting stimulus.

Characterization of radiation-induced thymine-tyrosine crosslinks by electrospray ionization mass spectrometry.

Exposure to ionizing radiation leads to formation of covalent crosslinks between DNA and proteins. The nature, extent and site of the modifications are not well understood due to the difficulty in assessing free radical-induced damage in biopolymers. Electrospray ionization mass spectrometry (ESI-MS) permits direct analyses of intact oligopeptides, permitting characterization of the radiation-induced DNA-protein covalently crosslinked constituents. Our first application of this methodology to free radical-induced damage was in a model system where angiotensin, a small 10-amino acid peptide, is irradiated at various doses in the presence of excess thymine. The relative yield of crosslinks, which ranged from 0.1 to 15%, was linearly related to radiation dose for doses from 0.1 to 100 Gy. Detection of thymine-tyrosine moieties in this model system was possible at doses as low as 0.1 Gy with a signal-to-noise ratio of 4 to 1. ESI-MS revealed that the site of crosslink was located exclusively on the tyrosine residue as expected.

Dichloroacetic acid and trichloroacetic acid-induced DNA strand breaks are independent of peroxisome proliferation.

This study examined whether the induction of single strand breaks in hepatic DNA by dichloroacetic acid (DCA) and trichloroacetic acid (TCA) depends upon peroxisome proliferation. Male B6C3F1 mice were given a single oral dose of either DCA or TCA. At varying times, between 1 and 24 h after administration of the compounds, breaks in DNA were measured using an alkaline unwinding assay. Peroxisome proliferation was monitored at the same time intervals in a parallel experiment by measuring peroxisomal B-oxidation of [14C]palmitoyl-CoA in liver homogenates. Both DCA and TCA significantly increased breaks in DNA at 1, 2, and 4 h post-treatment, with a return to control levels after 8 h. No evidence for an increase in peroxisomal beta-oxidation was produced by either chemical up to 24 h after administration. In a separate experiment, mice were treated with DCA or TCA for 10 days and their livers examined for evidence of peroxisome proliferation. An increase in liver weight was observed, particularly with DCA. Both TCA and DCA increased peroxisomal beta-oxidation in liver homogenates, with TCA-treated animals showing more activity than those treated with DCA. Electron microscopy revealed that the number of peroxisomes were approximately the same in DCA- and TCA-treated animals. However, peroxisomes induced by DCA treatment frequently lacked nucleoid cores. These data indicate that peroxisomes induced by these compounds differ in their concentration of peroxisomal enzymes. Except for a slight hypertrophy, repeated doses of TCA do not produce significant degenerative changes in the liver of mice. Repeated doses of DCA produce multifocal, subcapsular necrotic regions, and a marked hypertrophic response in the liver. Mice treated with TCA for 10 days and sacrificed 24 h after the last dose did not display increased strand breaks in hepatic DNA. This indicates that peroxisomal proliferation does not contribute to the induction of DNA strand breaks.

Inhibition of benzo[a]pyrene-7,8-diol formation in vitro by complex organic mixtures.

Coal-derived complex organic mixtures [COM] with boiling points greater than or equal to 370 degrees C (greater than or equal to 700 degrees F) are known to inhibit both mouse skin tumor initiation by benzo[a]pyrene [BAP], and BAP-induced bacterial mutagenesis. We have examined the effects of 5 COM, with boiling points of 149-370 degrees C (300-700 degrees F), 370-398 degrees C (700-750 degrees F), 398-426 degrees C (750-800 degrees F), 426-454 degrees C (800-850 degrees F), and greater than 454 degrees C (greater than 850 degrees F), on both the rate and the route of BAP metabolism by rat liver homogenates in vitro. When co-metabolized in 40:1 excess with BAP, all of the COM reduced the rate of BAP metabolism. The 149-370 degrees C (300-700 degrees F) COM reduced the initial rate of BAP metabolism to 34% of the rate for BAP alone, while the four higher-boiling COM reduced it to 6.3-9.3% of the rate for BAP alone. In addition, the 2 highest-boiling COM (426-454 degrees C and greater than 454 degrees C boiling points) were found to reduce the percentage of BAP metabolized to BAP-7,8-diol, in comparison to incubations using BAP alone. The 370-398 degrees C and 398-426 degrees C COM did not alter the percentage of BAP metabolized to BAP-7,8-diol, while the 149-370 degrees C COM increased it. Both the general inhibition of BAP metabolism (by all of the COM), and the specific inhibition of BAP-7,8-diol formation (by the highest-boiling COM) may play a role in the inhibition of formation of BAP-induced skin tumors by these materials.

Cytochrome P450 2E1 is the primary enzyme responsible for low-dose carbon tetrachloride metabolism in human liver microsomes.

We examined which human CYP450 forms contribute to carbon tetrachloride (CCl(4)) bioactivation using hepatic microsomes, heterologously expressed enzymes, inhibitory antibodies and selective chemical inhibitors. CCl(4) metabolism was determined by measuring chloroform formation under anaerobic conditions. Pooled human microsomes metabolized CCl(4) with a K(m) of 57 microM and a V(max) of 2.3 nmol CHCl(3)/min/mg protein. Expressed CYP2E1 metabolized CCl(4) with a K(m) of 1.9 microM and a V(max) of 8.9 nmol CHCl(3)/min/nmol CYP2E1. At 17 microM CCl(4), a monoclonal CYP2E1 antibody inhibited 64, 74 and 83% of the total CCl(4) metabolism in three separate human microsomal samples, indicating that at low CCl(4) concentrations, CYP2E1 was the primary enzyme responsible for CCl(4) metabolism. At 530 microM CCl(4), anti-CYP2E1 inhibited 36, 51 and 75% of the total CCl(4) metabolism, suggesting that other CYP450s may have a significant role in CCl(4) metabolism at this concentration. Tests with expressed CYP2B6 and inhibitory CYP2B6 antibodies suggested that this form did not contribute significantly to CCl(4) metabolism. Effects of the CYP450 inhibitors alpha-naphthoflavone (CYP1A), sulfaphenazole (CYP2C9) and clotrimazole (CYP3A) were examined in the liver microsome sample that was inhibited only 36% by anti-CYP2E1 at 530 microM CCl(4). Clotrimazole inhibited CCl(4) metabolism by 23% but the other chemical inhibitors were without significant effect. Overall, these data suggest that CYP2E1 is the major human enzyme responsible for CCl(4) bioactivation at lower, environmentally relevant levels. At higher CCl(4) levels, CYP3A and possibly other CYP450 forms may contribute to CCl(4) metabolism.

Comparative metabolism of carbon tetrachloride in rats, mice, and hamsters using gas uptake and PBPK modeling.

No study has comprehensively compared the rate of metabolism of carbon tetrachloride (CCl4) across species. Therefore, the in vivo metabolism of CCl4 was evaluated using groups of male animals (F344 rats, B6C3F1 mice, and Syrian hamsters) exposed to 40-1800 ppm CCl4 in a closed, recirculating gas-uptake system. For each species, an optimal fit of the family of uptake curves was obtained by adjusting Michaelis-Menten metabolic constants Km (affinity) and Vmax (capacity) using a physiologically based pharmacokinetic (PBPK) model. The results show that the mouse has a slightly higher capacity and lower affinity for metabolizing CCl4 compared to the rat, while the hamster has a higher capacity and lower affinity than either rat or mouse. A comparison of the Vmax to Km ratio, normalized for milligrams of liver protein (L/h/mg) across species, indicates that hamsters metabolize more CCl4 than either rats or mice, and should be more susceptible to CCl4-induced hepatotoxicity. These species comparisons were evaluated against toxicokinetic studies conducted in animals exposed by nose-only inhalation to 20 ppm 14C-labeled CCl4 for 4 h. The toxicokinetic study results are consistent with the in vivo rates of metabolism, with rats eliminating less radioactivity associated with metabolism (14CO2 and urine/feces) and more radioactivity associated with the parent compound (radioactivity trapped on charcoal) compared to either hamsters or mice. The in vivo metabolic constants determined here, together with in vitro constants determined using rat, mouse, hamster, and human liver microsomes, were used to estimate human in vivo metabolic rates of 1.49 mg/h/kg body weight and 0.25 mg/L for Vmax and Km, respectively. Normalizing the rate of metabolism (Vmax/Km) by milligrams liver protein, the rate of metabolism of CCl4 differs across species, with hamster > mouse > rat > human.

The PPAR-Platelet Connection: Modulators of Inflammation and Potential Cardiovascular Effects.

Historically, platelets were viewed as simple anucleate cells responsible for initiating thrombosis and maintaining hemostasis, but clearly they are also key mediators of inflammation and immune cell activation. An emerging body of evidence links platelet function and thrombosis to vascular inflammation. peroxisome proliferator-activated receptors (PPARs) play a major role in modulating inflammation and, interestingly, PPARs (PPARbeta/delta and PPARgamma) were recently identified in platelets. Additionally, PPAR agonists attenuate platelet activation; an important discovery for two reasons. First, activated platelets are formidable antagonists that initiate and prolong a cascade of events that contribute to cardiovascular disease (CVD) progression. Dampening platelet release of proinflammatory mediators, including CD40 ligand (CD40L, CD154), is essential to hinder this cascade. Second, understanding the biologic importance of platelet PPARs and the mechanism(s) by which PPARs regulate platelet activation will be imperative in designing therapeutic strategies lacking the deleterious or unwanted side effects of current treatment options.

Alterations in cytochrome P-450 levels in adult rats following neonatal exposure to xenobiotics.

Neonatal exposure to certain xenobiotics has been shown to alter hepatic metabolism in adult rats in a manner that indicates long-term changes in enzyme regulation. Previously, we have observed changes in adult testosterone metabolism and in cytochrome P-450 (P-450) mRNA levels in animals neonatally exposed to phenobarbital (PB) or diethylstilbestrol (DES). In order to test for other enzyme alterations, we used Western blot procedures for specific P-450s to analyze hepatic microsomes from adult rats (24 wk old) that had been exposed neonatally to DES, PB, 7,12-dimethylbenz[a]anthracene (DMBA), or pregnenolone 16 alpha-carbonitrile (PCN). The most striking effects were observed in the DES-treated males: P-4502C6 and an immunologically similar protein were increased 60 and 90%, respectively, relative to control values, but P-4503A2 was decreased by 44%. No changes were observed in the DES-treated males in levels of P-4502E1, P-4502B, or the male-specific P-4502C13. Adult males neonatally treated with PB had 150% increase in levels of anti-P4502B-reactive protein without significant changes in the other enzymes. The DES- and DMBA-treated females had increased levels of the female-specific P-4502C12 of 38 and 48%, respectively, but no other observed alterations. The results confirm that neonatal exposure to DES or PB can cause alterations in adult hepatic cytochrome P-450 levels but show that these chemicals act on different enzymes. Neonatal DMBA resulted in changes in adult females similar to those produced by the synthetic estrogen DES, but did so at about two-thirds lower dose.

Neonatal exposure to xenobiotics alters adult hepatic protein kinase C alpha levels and testosterone metabolism: differential effects by diethylstilbestrol and phenobarbital.

Hepatic enzymes that metabolize endogenous and xenobiotic compounds have been shown to be altered in adult rats that had been exposed to xenobiotics as neonates. Protein kinase C (PKC) is important in intracellular signaling and has been implicated in the regulation of hepatic monooxygenases. Therefore, we examined the effects of neonatal exposure to diethylstilbestrol (DES) and phenobarbital (PB) on hepatic microsomal testosterone metabolism and on the alpha form of protein kinase C (PKC alpha) in adult rats. In adult males, neonatal exposure to DES altered adult testosterone metabolism such that 7 alpha-hydroxylation was increased by 58% but 2 alpha-, 16 alpha-, and 6 beta-hydroxylations and conversion to androstenedione were decreased 31-44%. In contrast, adult males neonatally exposed to PB showed increased (20-27%) testosterone 2 alpha- and 16 alpha-hydroxylations and androstenedione formation, but no effect was observed in the rate of 6 beta- or 7 alpha-hydroxylations. Western blot analyses indicated that cytosolic PKC alpha levels in male rats neonatally exposed to PB were decreased by approximately 63% relative to the vehicle control group but were not significantly altered in the DES males. The PKC alpha levels generally correlated (r = -.75) with 16 alpha-hydroxytestosterone formation in all samples. These results show that neonatal treatment with DES or PB differentially alters hepatic monooxygenase enzyme activities and PKC alpha levels in adult rats.

DNA damage can alter the stability of nucleosomes: effects are dependent on damage type.

We have investigated the effects of DNA damage by (+/-)-anti-benzo[a]pyrene diol epoxide (BPDE) and UV light on the formation of a positioned nucleosome in the Xenopus borealis 5S rRNA gene. Gel-shift analysis of the reconstituted products indicates that BPDE damage facilitates the formation of a nucleosome onto this sequence. Competitive reconstitution experiments show that average levels of 0.5, 0.9, and 2.1 BPDE adducts/146 bp of 5S DNA (i.e., the size of DNA associated with a nucleosome core particle) yield changes of -220, -290, and -540 cal/mol, respectively, in the free energy (delta G) of nucleosome formation. These values yield increases of core histone binding to 5S DNA (K(a)) of 1.4-, 1.6-, and 2.5-fold, compared with undamaged DNA. Conversely, irradiation with UV light decreases nucleosome formation. Irradiation at either 500 or 2500 J/m2 of UV light [0.6 and 0.8 cyclobutane pyrimidine dimer/146 bp (on average), respectively] results in respective changes of +130 and +250 cal/mol. This translates to decreases in core histone binding to irradiated 5S DNA (K(a)) of 1.2- and 1.5-fold compared with undamaged DNA. These results indicate that nucleosome stability can be markedly affected by the formation of certain DNA lesions. Such changes could have major effects on the kinetics of DNA processing events.

Ornithine decarboxylase induction by chemically complex liquids from two solvent refined coal processes.

Studies with a variety of chemically purified substances have suggested that induction of the enzyme ornithine decarboxylase (ODC) in mouse epidermal cells may be a reliable indicator of neoplastic transformation. In an effort to extend these observations on ODC to chemically complex materials, we examined ODC induction by carcinogenic and non-carcinogenic mixtures and compared these results with tumorigenicity data for these materials. For these studies several boiling range fractions and several solvent-derived subfractions from two solvent-refined coal processes (SRC-I and SRC-II) were evaluated for their ability to induce ODC. Single applications of heavy distillate (HD), the SRC-II high-boiling fraction and a potent mouse skin carcinogen, produced ODC induction kinetics which were similar to that for 12-O-tetradecanoylphorbol-13-acetate (TPA). Both HD and TPA stimulated maximal ODC activity 3-5 h after application, with epidermal ODC levels returning to basal levels within 12 h. The magnitude of ODC induction after multiple applications of HD was not as great as that observed for TPA. Single skin applications of TPA and HD also transiently elevated hepatic ODC levels 27- and 7-fold, respectively; however, liver ODC activity did not increase following multiple applications of either chemical. Further, ODC induction by HD was also dose-dependent. Relative to controls, single applications of HD and process solvent (boiling range greater than 250 degrees C) elevated ODC levels 145- to 205-fold, light distillate and light oil (boiling range less than 180 degrees C) increased ODC levels 23- to 32-fold, and middle distillate and wash solvent (boiling range 180-250 degrees C) stimulated less than 2- to 8-fold increases in ODC. Single applications of three solvent-derived subfractions of HD, which are complete carcinogens, induced 3- to 7-fold ODC elevations over background levels; multiple applications of two of these subfractions elevated ODC levels 10- to 22-fold. Of the complex mixtures evaluated during this study, all complete carcinogens induced ODC; however, the magnitude and temporal pattern of induction varied with the material tested.

Isolation and measurement of 15N2 from respiratory gases of animals administered 15N-labeled substances.

A method is described for collection of metabolic 15N2 from in vitro preparations or intact rats administered 15N-containing compounds. The methods enables routine collection and mass spectrometric measurement of as little as 10 mumol 15N2 respired by a rat over a 24-h period. A device is described that includes either an animal chamber or a tissue reaction vessel in a closed recycling atmosphere, with automatic O2 replenishment and removal of CO2 and water. It is capable of sustaining moderate vacuum and is coupled to a high-vacuum manifold designed to process the contained atmosphere and respiratory gases. The starting atmosphere is an 80:20 mix of sulfur hexafluoride and O2. Recovery of 15N2 gas from the system without an animal present was 101.3 +/- 5.75%. When 15N2 gas was very slowly infused iv into an animal, recovery was 89.1 +/- 5.38%. Use of the method in studies of the fate of [15N]hydrazine in rats indicated that about 15% of the administered hydrazine is rapidly converted to 15N2, followed by slower conversion of an additional 7-10% over the next several hours.

Differential sensitivities of murine melanocytes and melanoma cells to buthionine sulfoximine and anticancer drugs.

High levels of intracellular glutathione (GSH) may result in resistance of tumor cells to cytotoxic drugs. Because of the innate refractory nature of melanoma cells to chemotherapy, we have used a syngeneic murine system consisting of nontumorigenic Mel-ab melanocytes, tumorigenic H-ras-transformed melanocytes (C9.1), and the highly metastatic BL6 melanoma cells to examine the GSH content, glutathione S-transferase (GST) activity, and sensitivity to buthionine sulfoximine (BSO) and other cytotoxic drugs. Compared to the nontumorigenic melanocytes, both C9.1 and BL6 melanoma cells have nearly fivefold higher GSH content, and BL6 cells have increased GST activity. C9.1 and BL6 cells are more resistant to the cytotoxic effects of BCNU and adriamycin; however, the degrees of resistance do not reflect the increased GSH content in these cells. Pretreatment of BL6 melanoma cells with 50 microM BSO depleted over 90% of their GSH content and enhanced the growth-inhibitory effects of L-dopa methylester, BCNU, bleomycin, and dacarbazine. Exposure to BSO alone was not toxic to the tumor cells for up to 24 hr, but was significantly cytotoxic in the melanocytes after 9 hr. The sensitivity of these cells to BSO appears to depend on a critical level of GSH depletion which is not related to the initial GSH content. These studies suggest that the resistance of melanoma cells to cytotoxic drugs is only partially attributed to changes in the GSH system caused during cellular transformation.

Nucleosome structure modulates benzo[a]pyrenediol epoxide adduct formation.

We have studied the binding of a chemical carcinogen to DNA reconstituted with histone octamers to determine the effect that nucleosome structure has on covalent adduct formation. Reconstitution of a plasmid containing the somatic 5S rRNA gene from Xenopus borealis resulted in characteristic nucleosome structure, as determined by micrococcal nuclease digestion, shifted migration in agarose gels, and hydroxyl radical footprinting. Formation of covalent adducts by benzo[a]pyrenediol epoxide (BPDE) occurred initially at a slower rate in reconstituted DNA than in naked plasmid, but after 2 h the total adduction levels (adducts/plasmid) were equal in both samples. Analysis of adduction at the sequence level by primer extension indicated that, after a 2-h BPDE reaction, the degree of adduction within the 5S rRNA nucleosome was suppressed by approximately 50% compared to naked DNA. The rotational setting of the guanines on the helix did not explain the level of adduction observed, since guanines in close proximity to the histone core were equally susceptible to adduction as guanines on the outer nucleosome surface. At early reaction times with BPDE, however, sequences near the 5S nucleosome dyad, where known modulations in the minor groove width occur, were the least susceptible to adduction. These results indicate that the structural features of DNA assembled into nucleosomes contribute to the susceptibility of the DNA to modification by BPDE.

Lung development and postnatal survival for rats exposed in utero to a high-boiling coal liquid.

Previous studies performed in this laboratory indicated that exposure of rat fetuses to high-boiling coal liquids from 12-14 days of gestation (dg) induced a number of major malformations, including cleft palate, diaphragmatic hernia and small lungs. The study reported here was designed to determine postnatal viability and development of survivors following in utero exposure to Harmarville process solvent (HPS), a wide-boiling-range (150 to greater than 455 degrees C) coal liquid. For this study, 0.74 g kg-1 of the coal liquid was administered (by intragastric intubation) to rats from 12 to 14 dg. Offspring were evaluated for postnatal survival, growth and lung and thymus weights. Randomly selected pups from control and treated litters were killed and necropsied at 1, 3, 7 and 21 days postpartum. In addition, data for control pups were obtained at 0.25 and 0.5 days postpartum for comparison with body, lung and thymus weights of pups that died during this interval. Fifty-four per cent of the exposed pups and 9% of the control pups died between birth and 3 days postpartum. Of the treated pups that died, 10% (6/5; pups/litters) had cleft palate, 27% (17/9) had small lungs and 33% (21/8) had both cleft palate and small lungs. No gross malformations were observed in the remaining 30% of the dead pups. Microscopic examination of lungs from HPS-treated pups revealed no evident histological abnormalities. Body, lung and thymus weights for treated animals that died were significantly less than those of controls.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA polymerase, RNA polymerase and exonuclease activities on a DNA sequence modified by benzo[a]pyrene diolepoxide.

Adducts produced by modification of DNA with benzo[a]pyrene diolepoxide (BPDE) are known to inhibit both DNA and RNA synthesis. This phenomenon has been used as a method for determining the distribution of carcinogen binding within defined DNA sequences. A critical comparison of different enzyme activities on adducted DNA is needed, since different enzymes may process adducted DNA differently. Thus, we compared blocks in DNA polymerase activity with that of an RNA polymerase and with an exonuclease at single base resolution. BPDE adducts blocked the progression of cloned T7 DNA polymerase (Sequenase) in a dose-dependent manner. Although the majority of these blocks were at one base prior to adducted guanines, we also observed some blocks opposite specific guanines, suggesting that in some sequences the polymerase inserted a base opposite the modified guanine. Digestion with T4 DNA polymerase (3'----5') exonuclease activity was also blocked in BPDE-adducted DNA; however, fragments produced by blocks in T4 exonuclease migrated two or more bases longer than the corresponding guanine. Mapping of adduct distributions using both Sequenase and T4 exonuclease gave similar results, demonstrating that a long tract of guanines was preferentially modified, and within a polyguanine sequence, the 5' guanines were more heavily modified than the 3' guanines. Transcription of adducted DNA by SP6 RNA polymerase was also inhibited in a dose-dependent manner. However, adducted bases which posed strong blocks to the DNA polymerase were not always strong blocks to the RNA polymerase. Thus, in terms of adduct distribution, Sequenase and T4 exonuclease provided more consistent results than the RNA polymerase, since blockage of the RNA polymerase correlated poorly with guanines.