Ten-year outcome of early childhood traumatic brain injury: Diffusion tensor imaging of the ventral striatum in relation to executive functioning.

PRIMARY OBJECTIVE: The long-term effects of TBI on verbal fluency and related structures, as well as the relation between cognition and structural integrity, were evaluated. It was hypothesized that the group with TBI would evidence poorer performance on cognitive measures and a decrease in structural integrity. RESEARCH DESIGN: Between a paediatric group with TBI and a group of typically-developing children, the long-term effects of traumatic brain injury were investigated in relation to both structural integrity and cognition. Common metrics for diffusion tensor imaging (DTI) were used as indicators of white matter integrity. METHODS AND PROCEDURES: Using DTI, this study examined ventral striatum (VS) integrity in 21 patients aged 10-18 years sustaining moderate-to-severe traumatic brain injury (TBI) 5-15 years earlier and 16 demographically comparable subjects. All participants completed Delis-Kaplan Executive Functioning System (D-KEFS) sub-tests. MAIN OUTCOMES AND RESULTS: The group with TBI exhibited lower fractional anisotropy (FA) and executive functioning performance and higher apparent diffusion coefficient (ADC). DTI metrics correlated with D-KEFS performance (right VS FA with Inhibition errors, right VS ADC with Letter Fluency, left VS FA and ADC with Category Switching). CONCLUSIONS: TBI affects VS integrity, even in a chronic phase, and may contribute to executive functioning deficits.

Sulforaphane induces phase II detoxication enzymes in mouse skin and prevents mutagenesis induced by a mustard gas analog.

Mustard gas, used in chemical warfare since 1917, is a mutagenic and carcinogenic agent that produces severe dermal lesions for which there are no effective therapeutics; it is currently seen as a potential terrorist threat to civilian populations. Sulforaphane, found in cruciferous vegetables, is known to induce enzymes that detoxify compounds such as the sulfur mustards that react through electrophilic intermediates. Here, we observe that a single topical treatment with sulforaphane induces mouse epidermal levels of the regulatory subunit of glutamate-cysteine ligase, the rate-limiting enzyme in glutathione biosynthesis, and also increases epidermal levels of reduced glutathione. Furthermore, a glutathione S-transferase, GSTA4, is also induced in mouse skin by sulforaphane. In an in vivo model in which mice are given a single mutagenic application of the sulfur mustard analog 2-(chloroethyl) ethyl sulfide (CEES), we now show that therapeutic treatment with sulforaphane abolishes the CEES-induced increase in mutation frequency in the skin, measured four days after exposure. Sulforaphane, a natural product currently in clinical trials, shows promise as an effective therapeutic against mustard gas.

Communication of direction by the honey bee.

In the presence of controls for site- and path-specific odors, observer and food-source scents, Nasanov gland and alarm odors, visual cues, wind, and general site taxis, recruited bees were able to locate the food source indicated by the dances of returning foragers in preference to a food source located at an equal distance in the opposite direction. This was true even when foragers were simultaneously dancing to indicate two different stations. Recruitment in the absence of dancing was very low, while in the absence of foraging it was virtually zero. Thus, under the experimental conditions used, the directional information contained in the dance appears to have been communicated from forager to recruit and subsequently used by the recruit.

Interactions of benzo(a)pyrene diol-epoxides with linear and supercoiled DNA.

Previous spectroscopic studies of the major adduct formed by reaction of (+/-)-7r,8t-dihydroxy-9t, 10t-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE-I) with linear DNA have been interpreted to suggest that the adduct is not intercalated in the double helix. However, studies of the electrophoretic mobility of supercoiled DNA treated with BPDE-I suggest that the adduct is intercalated. To resolve these interpretations, we have studied the reaction of BPDE-I with supercoiled and linear DNA. The kinetics of DNA-catalyzed hydrolysis and of covalent binding are similar for the two DNAs; supercoiled DNA exhibits a 20% increase in the rate of hydrolysis of BPDE-I at low DNA concentration compared to linear DNA. Fluorescence excitation spectra and fluorescence quenching experiments provide no support for a model in which BPDE-I adducts are intercalated in supercoiled DNA. When deoxyribonucleoside adducts were analyzed by high-performance liquid chromatography, identical distributions of BPDE-I adducts were found for supercoiled and linear DNA. These data are consistent with a previously proposed model (Hogan, M. E., Dattagupta, N., and Whitlock, J.P., Jr. J. Biol. Chem., 256: 4504-4513, 1981; Taylor, E.R., Miller, K. J., and Bleyer, A. J. J. Biomol. Struct. Dyn., 1: 883-904, 1983), in which the major BPDE-I adduct in both linear and supercoiled DNA exists in a conformation which allows stacking with the neighboring base pair and introduces a "kink" into the path of the helical axis. Although this model provides an explanation for all available experimental data, there are undoubtedly other DNA adduct conformational models which are also consistent with the data.

Differences in the binding of stereoisomeric benzo(a)pyrene-7,8-diol-9,10-epoxides to histones in rat liver nuclei.

We have compared the covalent binding of two stereoisomeric benzo(a)pyrene-7,8-diol-9,10-epoxides to histones. From rat liver nuclei exposed to carcinogenic [3H]-(+/-)-7r,8t-dihydroxy-9t,10t-oxy-7,8,9,10-tetrahydrobenzo(a)p yrene [( 3H]BPDE-I) or noncarcinogenic [3H]-(+/-)-7r,8t-dihydroxy-9c,10c-oxy-7,8,9, 10-tetrahydrobenzo(a)pyrene [( 3H]BPDE-II), H1 and core histone fractions were prepared by differential acid extraction. The specific activity (dpm/mg protein) of the core histone fraction for [3H]BPDE-I was much higher than that of [3H]BPDE-II. Alternatively in the H1 histone fraction, the binding level of [3H]BPDE-I was lower than that of [3H]BPDE-II. By reverse-phase high performance liquid chromatography, the analyses of BPDE isomers binding to histones showed that histones H2A and H1 were heavily labeled by [3H]BPDE-I and -II, respectively. In particular, the ratio of specific activities for BPDE-I to II in peak C3, which mainly contains H2A X 2 variant, was higher than those of other histone H2A variants and other core histones. These results indicate that the BPDE isomers have differential binding affinities to histones. The covalent binding of BPDE-I to histone H2A (especially H2A X 2 variant) may be important in the potential carcinogenic effects in nuclei.

Inhibition by 6-mercaptopurine of the binding of a benzo(a)pyrene diol-epoxide to DNA in Chinese hamster ovary cells.

The finding that 7r,8t-dihydroxy-9,10-t-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE-I) is stabilized against hydrolysis by binding to cellular membranes suggested that nucleophilic compounds which would colocalize with BPDE-I in membranes might inhibit the deleterious biological effects of BPDE-I. We have explored the possibility that hydrophobic, sulfhydryl-containing compounds might provide such inhibition using the binding of BPDE-I to DNA in Chinese hamster ovary cells as a biological end point. Of several such compounds tested, 6-mercaptopurine (6-MP) was the most potent, exhibiting 50% inhibition of BPDE-I:DNA binding at about 30 microM and about 95% inhibition at 500 microM. 6-MP, at concentrations of 30 microM or greater, was also effective in preventing the induction of mutations by BPDE-I at the aprt locus. By varying the time of addition of the two compounds, it was shown that the action of 6-MP is intracellular. In vitro, 6-MP readily forms an adduct with BPDE-I, and the same adduct is found as a major metabolite in cells treated with BPDE-I and 6-MP. These findings are consistent with the hypothesis that 6-MP and BPDE-I colocalize in membranes of Chinese hamster ovary cells and form a covalent adduct, thus preventing the BPDE-I from interacting with critical cellular macromolecules such as DNA. Several nontoxic derivatives of 6-MP (9-methyl-6-MP, 2,6-dithiopurine) or analogues of 6-MP (4-mercapto-1H-pyrazolo[3,4-d]pyrimidine) were also tested in the Chinese hamster ovary cell system and found to inhibit binding of BPDE-I to DNA with potencies comparable to that of 6-MP.

Metabolism and macromolecular binding of carcinogenic and noncarcinogenic metabolites of benzo(a)pyrene by hamster embryo cells.

The metabolism and macromolecular binding of four metabolites of benzo(a)pyrene in hamster embryo fibroblasts has been studied. Two noncarcinogenic phenolic derivatives, 3-hydroxybenz(a)pyrene and 9-hydroxybenzo(a)pyrene, are rapidly metabolized, primarily to their respective glucuronic acid conjugates and other H2O-soluble conjugates (78.4 to 80.8% of total radioactivity). Water-soluble conjugates were also formed from the carcinogenic phenol, 2-hydroxybenzo(a)pyrene, and from 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene, but in lower amounts (36.8 to 43.8% of total radioactivity. With each of the compounds, from 10 to 20% of the radioactivity was converted to ethyl acetate-soluble metabolites. The amount of unmetabolized 2-hydroxybenzo(a)pyrene recovered intracellularly was 20-fold higher than that recovered in incubations with the other phenols. Covalent binding to nuclear macromolecules was monitored after isopyknic separation. Binding of the three phenols tested was similar and was lower than the binding of benzo(a)pyrene to nuclear DNA, RNA, and protein. In contrast to the results with the monohydroxybenzo(a)pyrenes, high levels of covalent binding were observed with 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene; binding to DNA was 8-fold higher (315 pmol bound per mg DNA) than binding of benzo(a)pyrene to DNA.

Inhibition by 2,6-dithiopurine and thiopurinol of binding of a benzo(a)pyrene diol epoxide to DNA in mouse epidermis and of the initiation phase of two-stage tumorigenesis.

The chemotherapeutic agent 6-mercaptopurine was previously shown to inhibit the binding of 7r,8t-dihydroxy-9,10t-oxy-7,8,9,10-tetrahydro-benzo(a) pyrene (BPDE-I) to DNA in Chinese hamster ovary cells. Two compounds related to 6-mercaptopurine, 2,6-dithiopurine (DTP) and thiopurinol (TP), have been tested for inhibition of the binding of BPDE-I to epidermal DNA in mouse skin. Doses of test compound (0.2-20 mumol) or solvent control were applied to the shaved backs of female SENCAR mice. Fifteen min later, 200 nmol [3H]BPDE-I were applied to the same area and 3 h later the mice were sacrificed and epidermal DNA was purified and adduct formation was quantitated radiometrically. At the highest doses studied, DTP and TP inhibited DNA binding by 90 and greater than 80%, respectively. The dose necessary to inhibit DNA binding by 50% was about 0.8 mumol for DTP and about 2 mumol for TP. To test whether this protective effect was long-lasting, the time between application of purinethiol and [3H]BPDE-I was systematically increased. Although the level of protection was decreased by increasing the time between applications, both compounds inhibited binding 50-60% even after 24-48 h. A radioactive compound tentatively identified as a TP-BPDE-I adduct could be recovered from epidermal homogenates following topical application of TP and BPDE-I. We used a standard two-stage initiation-promotion protocol to test the effects of these compounds on mouse skin carcinogenesis. Mice were treated with 0, 1, or 10 mumol of either TP or DTP, and 15 min later were treated with an initiating dose of BPDE-I (200 nmol). Twice weekly promotion with 12-O-tetradecanoylphorbol-13-acetate was begun 2 weeks later and continued for 23 weeks. A dose-dependent inhibition of tumor incidence and multiplicity was noted with both compounds. Treatment of skin with 10 mumol of DTP prior to initiation lowered the number of papillomas per mouse by greater than 90% compared to solvent controls; a 10-fold lower dose resulted in about 50% inhibition. The 10-mumol dose of TP resulted in about 50% inhibition. Mice were examined for 50 weeks for the presence of squamous cell carcinomas. Compared to the positive control group, 10 mumol DTP inhibited carcinoma incidence and lowered the total number of carcinomas by 90-95%. Treatment with 10 mumol TP had no significant effect on carcinoma incidence, and only slightly lowered the total number of carcinomas.

Binding of benzo(a)pyrene derivatives to specific proteins in nuclei of intact hamster embryo cells.

In hamster embryo cells incubated for 24 hr with 4 microM [3H]benzo(a)pyrene, a major portion of the nonextractable radioactivity in nuclear preparations copurifies with the protein fraction. When these proteins are analyzed by sodium dodecyl sulfate:polyacrylamide gel electrophoresis, significant variations in the labeling intensities of the various proteins are seen. Control experiments demonstrate that the labeling is due to covalent binding to protein. Histones H3 an H2A are heavily labeled while the other histones of the nucleosome core, H2B and H4, are devoid of radioactivity. Large amounts of label are associated with proteins with mobilities similar to the very lysine-rich histones H1. However, the results of differential extraction experiments suggest that the labeled proteins do not belong to either the H1 or the high-mobility-group class of chromosomal proteins. During 6 hr of inhibition of protein synthesis by cycloheximide, the metabolism of [3H]benzo(a)pyrene, as monitored by high-pressure liquid chromatography, remained normal. Patterns of labelling of nuclear proteins after 3 or 6 hr were identical in the presence and absence of cycloheximide. This finding strongly suggests that binding of benzo(a)pyrene derivatives to nuclear proteins occurs in situ.

Effects of estrogen on global gene expression: identification of novel targets of estrogen action.

The important role played by the sex hormone estrogen in disease and physiological processes has been well documented. However, the mechanisms by which this hormone elicits many of its normal as well as pathological effects are unclear. To identify both known and unknown genes that are regulated by or associated with estrogen action, we performed serial analysis of gene expression on estrogen-responsive breast cancer cells after exposure to this hormone. We examined approximately 190,000 mRNA transcripts and monitored the expression behavior of 12,550 genes. Expression levels for the vast majority of those transcripts were observed to remain constant upon 17beta estradiol (E2) treatment. Only approximately 0.4% of the genes showed an increase in expression of > or =3-fold by 3 h post-E2 treatment. We cloned five novel genes (E2IG1-5), which were observed up-regulated by the hormonal treatment. Of these the most highly induced transcript, E2IG1, appears to be a novel member of the family of small heat shock proteins. The E2IG4 gene is a new member of the large family of leucine-rich repeat-containing proteins. On the basis of architectural and domain homology, this gene appears to be a good candidate for secretion in the extracellular environment and, therefore, may play a role in breast tissue remodeling and/or epithelium-stroma interactions. Several interesting genes with a potential role in the regulation of cell cycle progression were also identified to increase in expression, including Pescadillo and chaperonin CCT2. Two putative paracrine/autocrine factors of potential importance in the regulation of the growth of breast cancer cells were identified to be highly up-regulated by E2: stanniocalcin 2, a calcium/phosphate homeostatic hormone; and inhibin-beta B, a TGF-beta-like factor. Interestingly, we also determined that E2IG1 and stanniocalcin 2 were exclusively overexpressed in estrogen-receptor-positive breast cancer lines, and thus they have the potential to serve as breast cancer biomarkers. This data provides a comprehensive view of the changes induced by E2 on the transcriptional program of human E2-responsive cells, and it also identifies novel and previously unsuspected gene targets whose expression is affected by this hormone.

Geographic, demographic, and socioeconomic variations in the investigation and management of coronary heart disease in Scotland.

OBJECTIVE: To determine whether age, sex, level of deprivation, and area of residence affect the likelihood of investigation and treatment of patients with coronary heart disease. DESIGN, PATIENTS, AND INTERVENTIONS: Routine discharge data were used to identify patients admitted with acute myocardial infarction (AMI) between 1991 and 1993 inclusive. Record linkage provided the proportion undergoing angiography, percutaneous transluminal coronary angioplasty (PTCA), and coronary artery bypass grafting (CABG) over the following two years. Multiple logistic regression analysis was used to determine whether age, sex, deprivation, and area of residence were independently associated with progression to investigation and revascularisation. SETTING: Mainland Scotland 1991 to 1995 inclusive. MAIN OUTCOME MEASURES: Two year incidence of angiography, PTCA, and CABG. Results-36 838 patients were admitted with AMI. 4831 (13%) underwent angiography, 587 (2%) PTCA, and 1825 (5%) CABG. Women were significantly less likely to undergo angiography (p < 0.001) and CABG (p < 0.001) but more likely to undergo PTCA (p < 0.05). Older patients were less likely to undergo all three procedures (p < 0.001). Socioeconomic deprivation was associated with a reduced likelihood of both angiography and CABG (p < 0.001). There were significant geographic variations in all three modalities (p < 0.001). CONCLUSION: Variations in investigation and management were demonstrated by age, sex, geography, and socioeconomic deprivation. These are unlikely to be accounted for by differences in need; differences in clinical practice are, therefore, likely.

Mixed effects of 2,6-dithiopurine against cyclophosphamide mediated bladder and lung toxicity in mice.

2,6-Dithiopurine (DTP) has been proposed as a possible chemopreventive agent because of its ability to react with electrophiles. Acrolein, an electrophilic metabolite of cyclophosphamide (CP) involved in the toxicities of this anticancer drug, can be scavenged by DTP. The present study examined the effect of DTP treatment on CP-mediated bladder and lung toxicity in male ICR mice. Mice fed a diet containing 4% DTP that were treated intraperitoneally (i.p.) with 350 mg/kg CP showed no significant bladder damage (measured as bladder blood content at 48 h) with respect to the group fed a control diet. DTP (50 and 100 mg/kg), given i.p. 0.5 and 7 h after the initial injection of CP, also prevented the bladder damage when compared with the group receiving CP alone. Surprisingly, although neither parenteral CP nor DTP alone caused any mortality at these doses, the combined treatment resulted in 67% mortality within 3 days. At 24 h after CP + DTP, blood urea nitrogen was elevated 6-fold and urine volumes decreased by 70%. Histopathological analyses revealed a diffuse myocardial degeneration and necrosis, severe granular degeneration in the liver, abundant cellularity and infiltrates in interalveolar spaces in the lung and swollen nephron epithelial cells with some necrosis. All mice survived treatment when the dose of CP was lowered to 250 and 25-75 mg/kg DTP was given i.p. 0.5 and 7 h after CP. These DTP regimens reduced the degree of CP-induced lung toxicity, measured by [3H]thymidine incorporation into lung DNA 7 days after CP, in a dose-dependent manner. DTP (75 mg/kg) also reduced CP-induced lung fibrosis estimated by lung hydroxyproline content 28 days after CP. Analyses of urine from mice given CP + DTP revealed large amounts of the metabolic product dithiouric acid, smaller amounts of the parent DTP and several smaller peaks. The major unique metabolite peak was collected and analyzed by mass spectrometry, but did not correspond to either acrolein-DTP or acrolein-dithiouric acid. Thus, either very small amounts of an acrolein adduct are generated, the adduct is broken down to an unidentified product, or the ability of DTP to prevent CP-induced lung and bladder damage is related to some other mechanism. The possibility that mercapturic acid metabolites of acrolein released the parent electrophile in the urine was not supported by the finding that probenecid did not prevent CP-induced bladder toxicity.

Catalysis of carcinogen-detoxification by DNA: comparison of enantiomeric diol epoxides.

The interactions of the (+)- and (-)-enantiomers of 7r,8t-dihydroxy-9t,10t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I) with purified DNA have been studied in vitro. These compounds are formed by cellular metabolism of the potent environmental carcinogen benzo[a]pyrene, and the (+)-enantiomer is thought to be the ultimate carcinogenic metabolite. Non-covalent, intercalative binding was measured spectrophotometrically, hydrolysis was measured spectrofluorometrically and covalent binding was detected by liquid scintillation counting. No significant differences were found in the association constants for intercalative binding or in the ability of DNA to catalyse the hydrolysis of the two enantiomers. Covalent DNA binding was 4.5-fold higher for the (+)-enantiomer. When DNA was pretreated with a molar equivalent of the (-)-enantiomer, its subsequent ability to enhance the rate of BPDE-I hydrolysis and to bind covalently to (+)-BPDE-I was unimpaired. This suggests that the participation of the DNA in the hydrolysis reaction does not alter the DNA and therefore that the rate-enhancement is true catalysis.

Time course of metabolism of benzo[e]pyrene by hamster embryo cells and the effect of chemical modifiers.

In cultures of hamster embryo cells, benzo[a]pyrene (B[a]P) is metabolized primarily in the bay region. In contrast, little or no bay region metabolism of the noncarcinogenic isomer benzo[e]pyrene (B[e]P) could be detected during 12--96-h incubations of hamster embryo cells with 4 microM [3H]B[e]P. The upper limit to 9,10-dihydro-9,10-dihydroxy-B[e]P formation is about 0.2% of the ethyl acetate-soluble metabolites (less than 0.1% of the total metabolites). The major identified metabolites of B[e]P were 4,5-dihydro-4,5-dihydroxy B[e]P and the glucuronide conjugates of 3-OH-B[e]P and 4,5-dihydro-4,5-dihydroxy B[e]P. Simultaneous treatment of cells with either B[a]P or 7,8-benzoflavone (BF) did not induce bay region metabolism of [3H]B[e]P.

Stabilization of a reactive, electrophilic carcinogen, benzo[a]pyrene diol epoxide, by mammalian cells.

We have studied several features of the interactions of 7r,8t-dihydroxy-9t,10t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I) with a DNA repair-proficient derivative of Chinese hamster ovary cells (CHO), AT3-2, and with a UV-light sensitive mutant, UVL-10, derived from AT3-2. Methods were developed for quantitating the amount of unhydrolysed BPDE-I associated with cells and for purifying DNA from cells under conditions where artificial labeling during preparation is minimized. In both cell types, about 30% of the BPDE-I added to a cell culture is rapidly taken up by the cells and is maintained in a cellular compartment in which the half-life of BPDE-I is about 10-fold longer than in aqueous medium. The kinetics of covalent binding to DNA were measured in both cell types and found to be described well by a single exponential process with a half-life of about 60 min. This is virtually identical to the half-life for intracellular hydrolysis of BPDE-I (57 min), consistent with the suggestion that this intracellular, relatively stable BPDE-I is responsible for binding.

Differential efficiency of mutagenesis at three genetic loci in CHO cells by a benzo[a]pyrene diol epoxide.

The formation of DNA adducts by the ultimate carcinogen 7r,8t-dihydroxy-9t,10t-oxy-7,8,9,10-tetrahydrobenzo[alpha]pyrene (BPDE-I) has been implicated in the process of carcinogenesis. In a line of Chinese hamster ovary (CHO) cells designated AT3-2 and in two derivative mutant lines, UVL-1 and UVL-10, originally selected for hypersensitivity to UV-irradiation, we have measured the formation of BPDE-I: DNA adducts and the production of biological damage. The quantity and quality of BPDE-I: DNA adducts formed initially in the 3 cell lines are identical over a wide range of BPDE-I doses. However, the UVL lines are unable to remove adducts from their DNA, while the AT3-2 cells remove about 50% of the BPDE-I: DNA adducts in a 24-h incubation. Correlated with this, the UVL lines are more sensitive to the lethal effects of BPDE-I than are the AT3-2 cells. Mutant frequencies were measured at the aprt, hprt and oua loci and were found to increase linearly with BPDE-I: DNA adduct formation at doses which gave greater than 50% survival. At the hprt and oua loci, the efficiency of mutation induction was similar for AT3-2 and UVL-10 cells. UVL-1 cells showed slightly higher (within a factor of 2-3) mutant frequencies in response to BPDE-I compared to AT3-2 at these two loci. However, at the aprt locus the repair-deficient cells were much more highly mutable (9-15-fold) than the repair-proficient AT3-2 cells. Based on the measured average level of adduct formation, it is calculated that 15% of the BPDE-I: DNA adducts in the aprt gene are converted into mutations. However, the possibility exists that the aprt locus is subject to higher levels of modification by BPDE-I than is the bulk DNA, which would lead to an artifactually high apparent conversion frequency.

Differences in the rate of DNA adduct removal and the efficiency of mutagenesis for two benzo[a]pyrene diol epoxides in CHO cells.

The initiation of carcinogenesis by carcinogens such as 7r,8t-dihydroxy-9,10t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I) is thought to involve the formation of DNA adducts. However, the diastereomeric diol epoxide, 7r,8t-dihydroxy-9,10c-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-II), also forms DNA adducts but is inactive in standard carcinogenesis models. We have measured the formation and loss of DNA adducts derived from BPDE-II in a DNA-repair-proficient line of Chinese hamster ovary (CHO) cells, AT3-2, and in two derived mutant cell lines, UVL-1 and UVL-10, which are unable to repair bulky DNA adducts. BPDE-II adducts were lost from cellular DNA in AT3-2 cells with a half-life of 13.8 h; this was about twice the rate found for BPDE-I adducts. BPDE-II adducts were also lost from DNA in UVL-1 and UVL-10 cells, but at a much slower rate. When purified DNA was modified in vitro with BPDE-II and then held at 37 degrees C, DNA adducts were removed at a rate identical to that seen in UVL-1 and UVL-10 cells, suggesting that the loss in these cells was not due to enzymatic DNA-repair processes but to chemical lability of the adducts. Mutant frequencies at the APRT and HPRT loci were measured at BPDE-II doses that resulted in greater than 20% survival, and were found to increase linearly with dose. In the DNA-repair-deficient cells, the HPRT locus was moderately hypermutable compared with AT3-2 cells (about 5-fold); the APRT locus was extremely hypermutable, giving about 25-fold higher mutant fractions in UVL-1 and UVL-10 than in AT3-2 cells at equal initial levels of binding. When we compared the mutational efficiency of BPDE-II at both loci in AT3-2 cells (the mutant frequency in mutants/10(6) survivors at a dose that resulted in one adduct per 10(6) base pairs) with our previous studies of BPDE-1, we found that BPDE-II was 4-5 times less efficient as a mutagen than BPDE-I. This difference in mutational efficiency could be explained in part by the increased rate of loss of BPDE-II adducts from the cellular DNA, part of which was due to an increased rate of enzymatic removal of these lesions compared with the removal of BPDE-I adducts.

Low absolute mutagenic efficiency but high cytotoxicity of a non-bay region diol epoxide derived from benzo[a]pyrene.

Insights into the mechanisms of chemical carcinogenesis can sometimes be gained by comparing the effects of closely related chemicals which differ in carcinogenic potency. We have treated Chinese hamster ovary (CHO) cells with a non-carcinogenic metabolite of benzo[a]pyrene, 9r,10t-dihydroxy-7c,8c-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-III), and measured the formation and persistence of DNA adducts. We have correlated this binding data with cytotoxicity and mutagenicity in a DNA-repair-proficient CHO cell line (AT3-2) and in two derived lines, UVL-1 and UVL-10, which are unable to repair bulky DNA adducts. These data are compared with similar studies of the effects of the carcinogenic metabolite, 7r,8t-dihydroxy-9t,10t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I). Synchronous fluorescence spectroscopy was used to measure the levels of BPDE-III-DNA adducts in treated cells. Adduct levels increased linearly with dose, but the absolute binding levels were about 30-fold lower than in comparable incubations with BPDE-I. Measurements of the removal of adducts derived from these two diol epoxides indicated no significant difference in the rate of repair measured 24 h post-treatment. When cells were treated with increasing doses of BPDE-III, survival curves were obtained which exhibited a shoulder region at low doses and an exponential decrease in plating efficiency at higher doses. By comparison of the D0's, the DNA-repair-deficient cell lines were found to be 4-5-fold more sensitive to the killing effects of BPDE-III than were the repair-proficient AT3-2 cells.

DNA sequence and nucleosome placement on the murine fibroblast growth factor-4 gene.

A large portion of the 5' flanking region of the fibroblast growth factor-4 (FGF-4) gene has been sequenced, but only 38 bp of sequence information past the end of the last exon of this gene has been described. In this study, a total of 2769 bp of nucleotide sequence down-stream of the last exon of FGF-4 (GenBank #U43515) was determined by a combination of deletional subcloning and primer walking. In addition, we have used the theoretical algorithm of Calladine and Drew to predict the rotational positioning of nucleosomes throughout the entire FGF-4 gene.