Myeloperoxidase--463A variant reduces benzo[a]pyrene diol epoxide DNA adducts in skin of coal tar treated patients.

The skin of atopic dermatitis patients provides an excellent model to study the role of inflammation in benzo[a]pyrene (BaP) activation, since these individuals are often topically treated with ointments containing high concentrations of BaP. In this study we have determined, by HPLC with fluorescence detection, the BaP diol epoxide (BPDE)-DNA adduct levels in human skin after topical treatment with coal tar and their modulation by the -463G-->A myeloperoxidase (MPO) polymorphism, which reduces MPO mRNA expression. BPDE-DNA adduct levels were 2.2 and 14.2 adducts/10(8) nt for MPO-463AA/AG and -463GG, respectively. The predominant BaP tetrol observed was tetrol I-1, which is derived after hydrolysis of the anti-BPDE-DNA adduct. The tetrol I-1/II-2 ratio, corresponding to the anti/syn ratio, was 6.7. The (32)P-post-labeling assay was also performed and thin layer chromatograms showed a major spot with a chromatographic location corresponding to BPDE-DNA. The mean values of the BPDE-DNA adduct spots were 3.8 +/- 2.4 per 10(8) nt for MPO-463AA/AG (n = 3) and 18.4 +/- 11.0 per 10(8) nt for MPO-463GG (n = 7), respectively (P = 0.03). One individual with the homozygous mutant genotype (-463AA) even had a 13-fold lower adduct level (1.4 per 10(8) nt) as compared to MPO-463GG subjects. In conclusion, these data show for the first time: (i) the in vivo formation of BPDE-DNA adducts in human skin treated with coal tar; (ii) that the MPO-463AA/AG genotype reduced BPDE-DNA adduct levels in human skin.

Modulation of benzo[a]pyrene diolepoxide-DNA adduct levels in human white blood cells by CYP1A1, GSTM1 and GSTT1 polymorphism.

The modulation of benzo[a]pyrene diolepoxide (BPDE)-DNA adduct levels by polymorphisms in the CYP1A1, GSTM1 and GSTT1 genes was assessed in leukocytes of Caucasian males. Eighty-nine coke oven workers (35 smokers, 36 ex-smokers and 18 non-smokers) were recruited from job categories with different exposure levels to polycyclic aromatic hydrocarbons (PAH), together with 44 power plant workers (all smokers) not exposed to PAH. BPDE-DNA adducts were detected in 69 of 133 (52%) DNA samples with a 100-fold variation (range 0.2-44 adducts/10(8) nt) and a median of 1.6 adducts/10(8) nt. All samples with the GSTM1 active genotype (n = 59) and five out of 74 samples with GSTM1*0/*0 (7%) showed non-detectable adducts (<0.2 adducts/10(8) nt) and 69 of 74 subjects with GSTM1*0/*0 (93%) had detectable adducts (>0.2 adducts/10(8) nt). The difference in adduct level between the GSTM1*0/*0 and GSTM1 active genotypes was highly significant (P < 0.0001). No significant difference in adduct level between the GSTT1*0/*0 and GSTT1 active genotypes was seen. All heterozygotes (CYP1A1*1/*2) from subjects of GSTM1 active type did not have detectable adducts. Among the GSTM1-deficient individuals (n = 69), 42 with the CYP1A1*1/*1 genotype showed a lower adduct level (median 1.3, range 0.2-4.1 adducts/10(8) nt) compared with 26 individuals with heterozygous mutated CYP1A1*1/*2 genotypes (median 2.5, range 0.4-6.1 adducts/10(8) nt, P < 0.015). One individual with low PAH exposure and the rare combination CYP1A1*2A/*2A-GSTM1*0/*0 showed an extremely high level of 44 adducts/10(8) nt. Significant differences in detectable adduct levels were found between the CYP1A1*1/*1 and CYP1A1*1/*2 genotypes in the exposed group low + medium (P = 0.01) and for all adduct levels, detectable and non-detectable (set at a fixed value), in highly exposed individuals and in ex-smokers (P = 0.03), whereas no such differences were observed in the control group. Mutated CYP1A1*1/*2 increased the adduct level in non-smokers from the exposed group (1.4 versus 2.2 adducts/10(8) nt), but had no effect on the smokers from the exposed group (2.3 versus 2.8 adducts/10(8) nt). When all variables were dichotomized, statistical evaluation showed that CYP1A1 status (P = 0.015), PAH exposure (P = 0.003) and smoking (P = 0.006) had significant effects on adduct levels which increased in the order: CYP1A1*1/*1 < CYP1A1(*1/*2 or *2A/*2A); environmental exposure < occupational exposure; non-smokers < smokers, whereby adducts increased with cigarette dose and the duration of smoking. Higher levels of BPDE-DNA adducts in individuals with the combined CYP1A1(1/*2 or *2A/*2A)-GSTM1*0/*0 genotype suggest that these genotype combinations are at increased risk for contracting lung cancer when exposed to PAH.

Polycyclic aromatic hydrocarbon-DNA adducts in humans: relevance as biomarkers for exposure and cancer risk.

The methodology applied for DNA adducts in humans has become more reliable in recent years, allowing to detect even background carcinogenic adduct levels in environmentally exposed persons. Particularly, combinations of the various methods now allow the elucidation of specific adduct structures with detection limits of 1 adduct in 108 unmodified nucleotides or even lower. The quantification of polycyclic aromatic hydrocarbon-DNA (PAH-DNA) adducts in human tissues and cells has been achieved with a number of highly sensitive techniques: immunoassays and immunocytochemistry using polyclonal or monoclonal antisera specific for DNA adducts or modified DNA, the assay, and adduct identification using physicochemical instrumentation. The results summarized in this review show that PAH-DNA adducts have been detected in a variety of human tissues, including target organs of PAH- and tobacco-associated cancers. Although dosimetry has not always been precise, a large number of data now clearly show that lowering exposure to carcinogenic PAH results in decreasing PAH-DNA adduct levels. In most studies, however, bulk DNA of a certain tissue or cell type has been examined, and there were relatively few studies in which mutations as a consequence of DNA damage at specific genes have been investigated. Promising as these biomarker studies seem for epidemiology and health surveillance, future biomonitoring and molecular epidemiological studies should be directed to combine several endpoint measurements: i.e., adduct formation (preferably at specific sites), mutational spectra in cancer-relevant genes, and genetic markers of (cancer) susceptibility in a number of cancer-predisposing genes.

32P-postlabelling of aromatic DNA adducts in white blood cells and alveolar macrophages of smokers: saturation at high exposures.

DNA adducts may serve as a molecular dosimeter of exposure to cigarette smoke-associated carcinogens such as polycyclic aromatic hydrocarbons (PAH). Target tissues for cigarette smoke-induced carcinogenesis are rarely accessible; therefore, peripheral blood cells or cells obtained by bronchoalveolar lavage (BAL) may be used as surrogate sources of exposed DNA. However, the relationship between cigarette smoke exposure and aromatic-DNA adducts in white blood cells and BAL cells is still unclear. In this study, we examined DNA adduct formation in lymphocytes and BAL cells in several populations of smoking individuals by means of 32P-postlabelling. Significant correlations between the amount of cigarettes smoked per day and the level of aromatic-DNA adducts were found in lymphocytes. In BAL cells, DNA adduct levels were associated with age (p = 0.05) and gender (p = 0.10) after adjustment for smoking behaviour. Adduct formation levelled off at higher exposure levels, suggesting less efficient adduct formation; decreases in the formation of adducts per unit of exposure were found in lymphocytes (r(s) = -0.80, p < 0.001) and BAL cells (r(s) = -0.72, p < 0.001). To assess intra-individual variation in adduct levels at constant smoking behaviour, sampling was repeated after a period of 2 and 6 months. In lymphocytes, repeated measurements with an interval of 2 months were highly correlated (r = 0.84, p = 0.009, n = 8), whereas repeated measurements with an interval of 6 months showed no correlation (r = 0.30, p = 0.27, n = 16). Repeated measurements in BAL cells showed a significant correlation after 6 months (r = 0.68, p = 0.03, n = 10). Furthermore, in a group of occupationally exposed aluminium workers, adduct levels in total white blood cells were correlated with the average concentrations of PAH in the ambient air of workers who smoked cigarettes, whereas in non-smokers, no such relationship was found. We conclude that cigarette smoking may directly or indirectly influence DNA adduct levels and saturation of DNA adduct formation may occur, leading to non-linear dose-response relationships.

Comparison of 32P-postlabeling and HPLC-FD analysis of DNA adducts in rats acutely exposed to benzo(a)pyrene.

DNA adduct analysis is often used for biomonitoring individuals exposed to polycyclic aromatic hydrocarbons (PAH). The 32P-postlabeling assay is routinely applied to study the formation of aromatic bulky adducts, but cannot positively identify individual adduct types. Recently, an HPLC assay with fluorescence detection (HPLC-FD) was developed which was sufficiently sensitive to detect adducts formed by benzo[a]pyrene (B[a]P) diolepoxide isomers [(+/-)anti- and (+/-)syn-BPDE] in occupationally exposed subjects (Rojas et al. Carcinogenesis, 16 (1995) 1373-1376). In this study, we compared both techniques using DNA samples of rats which were treated i.p. with B[a]P (10 mg/kg bw). The internal dose was assessed by measuring 3-OH-B[a]P excretion in urine. The detection limit of the HPLC-FD assay varied from 0.5 to 7.4 adducts per 10(8) nucleotides, while the detection limit of the 32P-postlabeling assay was around 1 adduct per 10(9) nucleotides. HPLC-FD analysis showed that BPDE-DNA adduct levels were highest in the heart, lung and liver respectively. The most predominant B[a]P-tetrol was the I-1 isomer, which derives from hydrolysis of the major reaction product of DNA and (+)-anti-BPDE. 32P-postlabeling analysis revealed an adduct spot that comigrated with a [3H]BPDE-DNA standard. The putative BPDE-DNA adduct levels were highest in heart followed by lung and liver and correlated significantly with tetrol I-1 levels determined by HPLC-FD (r = 0.72, P = 0.006). In samples in which both tetrol I-1 and II-2 were detected by means of HPLC-FD, this correlation was even better (r = 0.95, P = 0.01). Estimated half-lives of BPDE-DNA adducts were in the ranking order; heart, lung and liver for both techniques. By 32P-postlabeling, adducts other than BPDE-DNA were also found, resulting in highest total DNA adduct levels in the liver, heart and lung respectively. Furthermore, mean 24 h urinary excretion of 3-OH-B[a]P was related to BPDE-DNA adduct levels in lung, liver and heart. The 32P-postlabeling assay is sensitive and capable of detecting exposures to complex mixtures, whereas the HPLC-FD assay can be used to identify BPDE-isomers and might therefore be of value in risk assessment of individuals exposed to PAH.

Dopamine transporter immunoreactivity in rat brain.

The dopamine transporter (DAT) is a primary site for the action of cocaine in inducing euphoria. Its action is necessary for the selectivities of dopaminergic neurotoxins that provide the best current experimental models of Parkinson's disease. In the present report, rat dopamine transporter-like immunoreactivity (iDAT) was assessed by immunohistochemistry using newly developed polyclonal antisera raised against conjugated peptides corresponding to sequences found in the dopamine transporter's carboxy- and amino-termini. Dense iDAT was observed in patterns consistent with neural processes and terminals in the striatum, nucleus accumbens, olfactory tubercle, nigrostriatal bundle, and lateral habenula. Perikarya in the substantia nigra pars compacta were immunostained with moderate intensity using one of two immunohistochemical methods, while scattered ventral tegmental area perikarya were stained with somewhat less intensity. Immunoreactive neuronal processes with axonal and dendritic morphologies were stained in the substantia nigra and the paranigral and parabrachialis pigmentosus nuclei of the ventral tegmental area, while sparser processes were noted more medially in the ventral tegmental area. Neuronal processes were found in several laminae in the cingulate cortex, with notable fiber densities in the superficial aspects of lamina I and laminae II/III. The intensities of immunoreactivities in striatum and cerebral cortex were dramatically attenuated ipsilateral to nigrostriatal bundle 6-hydroxydopamine lesions. Specificity of immunostaining was supported by agreement of the results using sera directed against two distinct DAT segments, studies with preimmune and preadsorbed sera and studies of the extracted protein. These antisera identify and reveal details of the distribution of DAT immunoreactivity in rat brain and display variations in levels of DAT expression of likely functional significance.

Polycyclic aromatic hydrocarbon-DNA adducts in white blood cell DNA and 1-hydroxypyrene in the urine from aluminum workers: relation with job category and synergistic effect of smoking.

We examined a group of 105 workers from a primary aluminum plant for the presence of polycyclic aromatic hydrocarbon (PAH)-DNA adducts in their WBC and 1-hydroxypyrene in their urine. Workers were recruited from five job categories with different PAH exposure: the anode factory; the bake oven; and the electrolysis and the pot-relining departments. Unexposed workers from the foundry department served as the control group. The exposure to PAH was measured by personal monitoring, and the average PAH concentrations in the work atmosphere ranged from 0.4 micrograms/m3 in the foundry to 150 micrograms/m3 in the pot-relining department. The average exposure to benzo(a)pyrene was under the Swedish exposure limit of 5 micrograms/m3. The internal dose of pyrene was measured utilizing the 1-hydroxypyrene concentration in pre- and postshift urine samples. Higher exposure to PAH in the work atmosphere was associated with increased concentrations of 1-hydroxypyrene in the urine. The average increase in concentration of 1-hydroxypyrene ranged from 0.2 mumol/mol creatinine in the control group to 5.9 mumol/mol creatinine in the pot-relining department; an accumulation of 1-hydroxypyrene over a 5-day working period was observed. A good correlation was found between PAH exposure and the concentration of 1-hydroxypyrene in the urine on a group level (rs = 0.90; P = 0.02). PAH-DNA adducts were determined by 32P-postlabeling analysis (nuclease P1 enrichment procedure).(ABSTRACT TRUNCATED AT 250 WORDS)

Role of nucleotide excision repair in processing of O4-alkylthymines in human cells.

O4-Alkylthymines have been implicated as potential carcinogenic DNA lesions. We have studied the effects of O4-methylthymine, O4-ethylthymine, and O4-n-propylthymine in a model system in which a single lesion was located at a defined position on a SV40-based shuttle vector and have found large differences in the effects of these lesions in repair-proficient and nucleotide excision repair-deficient cells. In repair-competent human HeLa cells, normal fibroblasts, and XP-A (2OS) revertant cells, all 3 residues were highly mutagenic; a mutation frequency of approximately 20% was found for both O4-methylthymine and O4-ethylthymine, whereas that of O4-n-propylthymine was approximately 12%. These frequencies were independent of the activity of the O6-alkylguanine DNA alkyltransferase. All three O4-alkylthymines induced T-->C transitions exclusively. In nucleotide excision repair-deficient XP-A cells, however, these lesions were not mutagenic but strongly inhibited plasmid replication (> 90%). These results indicate that O4-alkylthymines are efficiently recognized by the nucleotide excision repair system and cause a complete cessation of plasmid replication if this system is deficient. Nevertheless, proficiency in the nucleotide excision repair pathway correlates with a high frequency of mutation induction by these lesions.

Validation of a new fluorometric assay for benzo[a]pyrene diolepoxide-DNA adducts in human white blood cells: comparisons with 32P-postlabeling and ELISA.

A new fluorometric assay was validated for quantification of benzo[a]pyrene diolepoxide (BPDE)-DNA adducts in white blood cells (WBC) from humans exposed to polycyclic aromatic hydrocarbons (PAH). This assay has a detection limit of 2 pg of r-7,c-10,t-8,t-9-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene derived from acid hydrolysis of BPDE-DNA, and can measure 1 BPDE adduct per 10(8) unmodified nucleotides. The quantity of WBC DNA required depends on the modification level and varies between 5 and 500 micrograms. The assay was applied to seven WBC DNA samples from lung cancer patients, six of whom were heavy smokers, and to three WBC DNA samples from healthy subjects employed in an aluminum production plant. High levels of BPDE-DNA adducts, ranging from 62 to 533 adducts/10(8) nucleotides were found in six out of seven DNA samples from the lung cancer patients. In WBC DNA from healthy persons BPDE-DNA adducts were detected only in two non-smokers, but at a much lower level than in lung cancer patients (4-10 adducts/10(8) nucleotides). Using coded WBC DNA samples, BPDE-DNA adduct levels measured by fluorometry of the B[a]P-tetrols, were compared with the results obtained by 32P-postlabeling (nuclease P1 enrichment) and ELISA measurements. A good correlation and proportionality was found between the levels of BPDE-DNA adducts measured by fluorometry and 32P-postlabeling (r = 0.95, P < 0.001, n = 8). The correlation between fluorometry and ELISA was much lower and not significant (r = 0.61, P = 0.1, n = 6). Moreover, the ELISA grossly overestimated BPDE-DNA adduct levels measured by the other two methods. The results demonstrate that the highly sensitive and specific fluorometric assay is suitable for measuring BPDE-DNA adducts in WBC from humans exposed to benzo[a]pyrene.

Sympathetic activity and presynaptic adrenoceptor function in patients with longstanding essential hypertension.

OBJECTIVE: To assess the contribution of the sympathetic nervous system to the hypertension in patients with longstanding essential hypertension. DESIGN: Overall sympathetic function, presynaptic adrenoceptors and neuronal re-uptake were examined after withdrawal of medication for at least 3 weeks in eight patients with longstanding essential hypertension and in eight carefully matched normotensive control subjects. METHODS: Minimal forearm vascular resistance after 10 min ischaemia was used as a measure of structural vascular changes. Overall sympathetic tone was assessed using tilt testing and pressor dose infusion of noradrenaline. The presence and function of presynaptic adrenoceptors and the neuronal re-uptake of noradrenaline were evaluated in the forearm using tracer noradrenaline kinetics with measurement of forearm noradrenaline plasma appearance rate and noradrenaline plasma spillover. Intra-arterial infusions of tritiated noradrenaline, the endogenous alpha- and beta-adrenoceptor agonist adrenaline, the alpha-adrenoceptor blocker phentolamine, the non-adrenergic vasodilator sodium nitroprusside and the neuronal re-uptake inhibitor desipramine were given in the forearm. RESULTS: We found that the hypertensives had higher minimal forearm vascular resistance, indicating structural vascular changes; decreased overall sympathetic activity, indicated by a lower basal whole-body noradrenaline production rate; enhanced vasopressor sensitivity for exogenously administered noradrenaline with decreased arterial baroreflex sensitivity; indications of decreased forearm neuronal re-uptake; evidence consistent with the presence of presynaptic, release-facilitating beta-adrenoceptors in the forearm, apparently not functionally different between the two groups; and undecisive evidence for the presence of functional presynaptic alpha-adrenoceptors in the forearm. CONCLUSIONS: In patients with longstanding essential hypertension we found decreased overall sympathetic activity, with indications of decreased forearm neuronal re-uptake, which might have a compensatory role. We found indications of structural vascular changes and diminished baroreflex sensitivity in the hypertensives, which contribute to the hypertension. However, peripheral presynaptic, release-facilitating beta-adrenoceptors seem to be present, which are functionally not clearly different between the two groups. Observations on peripheral presynaptic alpha-adrenoceptors were inconclusive.

Trained and amphetamine-induced circling behavior in lesioned, transplanted rats.

Rats were trained to turn for water reinforcement and then were given unilateral 6-hydroxydopamine lesions. After lesion, rats showed deficits in trained turning both contra- and ipsilateral to the side of the lesion, with contralateral turning more severely impaired. The lesioned rats were then transplanted with fetal mesencephalic dopamine tissue into striatum. A control group of lesioned rats were sham transplanted. Four weeks after transplant, 1.5 mg/kg D-amphetamine challenge injections were used to test the functioning of the transplants. In the control rats, D-amphetamine induced ipsilateral turning; in transplanted rats, D-amphetamine slowed the rate of ipsilateral turning or reversed the direction of amphetamine-induced rotation. Only rats which reversed their amphetamine-induced turn direction after transplant were used for the rest of the experiment. Trained turning was assessed at 4, 8, 12 and 16 weeks post transplant. Transplants did not improve learned performance at any time post transplant. When D-amphetamine was administered in conjunction with the trained turning sessions, a low dose (0.12 mg/kg) enhanced contralateral trained turn rates, without affecting ipsilateral turn rates. Higher doses of amphetamine reduced ipsilateral turn rate in the transplanted animals. The results of this study suggest that transplants alone do not reinstate performance of conditioned rotation.

DNA adducts as a measure of lung cancer risk in humans exposed to polycyclic aromatic hydrocarbons.

Workers in the coking, foundry, and aluminum industry can be exposed to high concentrations of polycyclic aromatic hydrocarbons (PAHs) and are at increased risk for lung cancer, as are cigarette smokers. In recent years several studies on workers in the foundry and coking industries have been reported. In these studies, white blood cell(WBC) DNA was used for analysis of PAH-DNA adducts. Theoretically, DNA adduct formation is a more relevant biological parameter for assessing exposure risk than PAH in the work atmosphere, or the amount of a metabolite in the urine, because adduct levels reflect that part of the dose that escapes detoxification and binds to DNA. We analyzed WBC DNA from coke-oven workers and from workers in an aluminum production plant and demonstrated the presence of PAH-DNA adducts. Forty-seven percent of the coke-oven workers had detectable levels of PAH-DNA adducts in their WBC compared with 27% of the controls (p < 0.05), measured with ELISA. In both groups, smokers had significantly higher levels of PAH-DNA adducts than did nonsmokers. In the aluminum workers, no PAH-DNA adducts were detected by ELISA, although the benzo[a]pyrene concentrations in the work atmosphere were comparable to those of the coke-oven workers. The more sensitive 32P-postlabeling assay showed the presence of PAH-DNA adducts in 91% of the aluminum workers. There was no correlation of WBC adduct levels with the concentration of PAH in the work atmosphere. Recently we showed that total PAH-DNA adduct levels in WBC from lung cancer patients were much higher than those generally found in healthy smokers.(ABSTRACT TRUNCATED AT 250 WORDS)

Survival of implanted fetal dopamine cells and neurologic improvement 12 to 46 months after transplantation for Parkinson's disease.

BACKGROUND AND METHODS: Patients with Parkinson's disease tend to have a reduced response to levodopa after 5 to 20 years of therapy, with "on-off" fluctuations consisting of dyskinesia alternating with immobility. In an effort to modify the motor disability of advanced Parkinson's disease, we implanted embryonic mesencephalic tissue containing dopamine cells into the caudate and putamen of seven patients. Two patients received unilateral grafts in the caudate and the putamen on the side opposite the side with worse symptoms. Five patients received bilateral grafts implanted in the putamen only. In six of the seven patients, the fetal tissue was obtained from a single embryo with a gestational age of seven to eight weeks. The tissue was injected by means of 10 to 14 needle passes. There were no surgical complications. Four of the seven patients underwent immunosuppression with cyclosporine and prednisone. RESULTS: All patients reported improvement according to the Activities of Daily Living Scale when in the on state 3 to 12 months after surgery (P < 0.01). Neurologic examination according to the Unified Disease Rating Scale showed that five of the seven patients improved when in the on state six months after surgery. The mean group Hoehn-Yahr score improved from 3.71 to 2.50 (P < 0.01). Computer and videotape testing in the home supported these findings. Fluctuations in clinical state were moderated, and periods of dyskinesia and off episodes were shorter and less severe than before implantation. Drug doses were reduced by an average of 39 percent (P < 0.01; maximum, 58 percent). The results of clinical evaluation and fluorodopa positron-emission tomography in one patient were compatible with transplant survival for as long as 46 months. Both immunosuppressed and nonimmunosuppressed patients improved. CONCLUSIONS: Fetal-tissue implants appear to offer long-term clinical benefit to some patients with advanced Parkinson's disease.

The electrocardiographic, clinical, and electrophysiologic spectrum of idiopathic monomorphic ventricular tachycardia.

Clinical, ECG, and electrophysiologic data from 47 patients who had episodes of sustained or nonsustained monomorphic VT with no evidence of structural heart disease were reviewed. According to the QRS configuration during tachycardia, four groups were distinguished. Nine patients had a right bundle branch block configuration and superior frontal plane QRS axis (group 1). Nine patients had a right bundle branch block configuration but an intermediate or right QRS axis (group 2). Group 3 consisted of five patients with a left bundle branch block configuration and a left axis deviation, and in group 4 there were 24 patients who had a left bundle branch block configuration with an intermediate or right frontal axis. Patients in group 1 had dizziness during tachycardia less frequently, but they needed cardioversion to terminate their arrhythmias more often. They experienced tachycardia during exercise less often, and tachycardia was not initiated during exercise testing. They had fewer ventricular premature beats according to the Holter recording. During the electrophysiologic study, VT was induced and terminated by pacing more often in this group. Patients with idiopathic VT with a right bundle branch block configuration and a superior axis seem to be a unique group of patients with idiopathic VT, and reentry seems to be the most likely arrhythmia mechanism in this group. The other ECG configurations share the same clinical and electrophysiologic characteristics, which suggest that the underlying arrhythmia mechanism is the same.

Repair and replication of plasmids with site-specific 8-oxodG and 8-AAFdG residues in normal and repair-deficient human cells.

The in vivo mutagenicity of 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) and N-(guanin-8-yl)-N-acetyl-2-aminofluorene (8-AAFdG) in human cells was determined by transfecting various cell lines with plasmids that carried a single adduct at a defined site. 8-OxodG is one of the many DNA modifications formed by oxygen radicals, and was found to be highly miscoding during replication with purified DNA polymerases in vitro. Here we show that the frequency of mutations induced by 8-oxodG during replication in vivo is at most only 2% above background. The most predominant mutation found was a single G----T transversion. The frequency of this transversion was found to be 3 to 5-fold increased in excision repair deficient XP-A cells. Interestingly, also the replication of 8-oxodG containing plasmids was significantly impaired (approximately 4-fold) in the XP-A cells, but not in HeLa cells, normal fibroblasts or XP-A revertant cells. When 8-AAFdG containing plasmids were used, the mutation frequencies did not exceed background levels (less than 2%) with any of the cell lines tested. The presence of 8-AAFdG almost completely inhibited plasmid replication (more than 50-fold) in XP-A cells. Apparently, both 8-AAFdG and 8-oxodG are not or poorly repaired in these cells, causing a block of DNA replication. This suggests that both lesions are substrates for excision repair, although to a varying extent.

Polycyclic aromatic hydrocarbon--DNA adducts in white blood cells from lung cancer patients: no correlation with adduct levels in lung.

Smokers of cigarettes are exposed to a number of carcinogens, including polycyclic aromatic hydrocarbons (PAHs), and are at a high risk for lung cancer. PAHs exert their carcinogenic activity after metabolic activation to reactive intermediates that can damage DNA through adduct formation. Measuring DNA adducts in peripheral white blood cells (WBC) could serve as a means of monitoring human exposure to genotoxic agents and subsequently risk assessment. In this study, DNA from WBC obtained from 39 lung cancer patients was examined for PAH-DNA adducts both in an ELISA using a polyclonal antibody against benzo[a]pyrene 7,8-diol-9,10-epoxide (BPDE)-DNA and the 32P-post-labeling technique. The ELISA results showed BPDE-DNA antigenicity in WBC DNA from 12/38 (32%) patients and adduct levels ranged from 1.5 to greater than 150 adducts in 10(8) nucleotides. The autoradiographs of chromatograms of 32P-post-labeled digests of WBC DNA from the 38 patients showed a variety of adduct spots; relative adduct labeling (RAL) values ranged from 0.3 to 407 adducts in 10(8) nucleotides. In 18 of the 38 (47%) persons an adduct spot was detected that co-chromatographed with the major BPDE-DNA adduct (BPDE-dG); RAL values ranged from 0.03 to 382 adducts in 10(8) nucleotides. Correlations were not significant between the adduct levels in WBC and smoking habits, age or sex. From 20 patients of the same group lung tissue was collected at surgery and examined for PAH-DNA adducts by ELISA and 32P-post-labeling assay. No significant correlation was found between DNA adduct levels in blood and lung. This finding stresses the limitations of the use of WBC as a surrogate for adduct levels in the target organ.

Detection of 8-hydroxyguanine in small amounts of DNA by 32P postlabeling.

A method for the sensitive detection of 8-hydroxyguanine residues in small amounts of DNA (0.2-2 micrograms) was developed. It comprises (i) the enzymatic hydrolysis of DNA to 2'-deoxyribonucleotide 3'-monophosphates, (ii) degradation of the bulk amount of normal purine and pyrimidine deoxyribonucleotides in the DNA digest by treatment with trifluoroacetic acid and hydrazine, respectively, under conditions retaining the structure of d(8-OH-G)p necessary for 5' phosphorylation by T4 polynucleotide kinase (PNK), (iii) 5' phosphorylation of d(8-OH-G)p by T4 PNK-catalyzed transfer of 32P from [gamma-32P]ATP, and (iv) 2D thin-layer chromatography on polyethyleneimine-cellulose sheets to purify and resolve 32P-postlabeled d(8-OH-G)p. Model experiments with mixtures composed of synthesized d(8-OH-G)p and DNA hydrolysate indicate that it is possible to detect one 8-hydroxyguanine residue out of 2 x 10(6) normal bases starting with 1 microgram DNA. The methodology, which allows for a further decrease of this detection limit, might be very useful for the sensitive detection of DNA damage induced by activated oxygen species in small amounts of DNA. We demonstrate the formation of 8-OH-G in DNA in vitro by low doses of 60Co gamma-rays.

Programmed electrical stimulation and drugs identify two subgroups of ventricular tachycardias occurring 16-24 hours after occlusion of the left anterior descending artery.

BACKGROUND: Spontaneous sustained ventricular tachycardia (VT) occurring 16-24 hours after left anterior descending (LAD) coronary artery occlusion in the canine heart is most likely based on abnormal automaticity. In vitro, it has been demonstrated that the rate of the arrhythmia and the effect of overdrive pacing depends on the maximal diastolic potential (MDP). The MDP is also of importance in understanding the effect of antiarrhythmic drugs. To study 1) the possible presence of different responses to overdrive pacing and 2) the relation between the response to overdrive pacing and the effect of different antiarrhythmic drugs in the intact heart, we investigated the effect of 1) (prolonged) pacing and 2) lidocaine (3 mg/kg), verapamil (0.4-1.0 mg/kg), or flunarizine (2 mg/kg) during VT. METHODS AND RESULTS: In 21 conscious dogs with chronic atrioventricular block, 60 sustained VTs were observed 1 day after LAD occlusion. During VT, pacing with interstimulus intervals of 400, 300, and 200 msec for 15, 60, and 120 seconds was done on 40 VTs. Based on their response to pacing, VTs were divided into a pacing-suppressible (PS group) and a pacing-nonsuppressible group (PNS group). The mean cycle length in the PS group was significantly longer (410 +/- 50 msec) than in the PNS group (360 +/- 35 msec, p less than or equal to 0.01). Suppression was directly related to the rate and duration of pacing. Spontaneous recurrence of VTs was observed after 26 +/- 45 seconds. Lidocaine and verapamil increased cycle length of the suppressible VTs and terminated them, whereas flunarizine had no effect. Except for verapamil, which increased cycle length of the VTs, no effects were seen in the PNS group. CONCLUSIONS: In conscious dogs showing sustained VTs 16-24 hours after LAD occlusion, 1) the slower VTs can be suppressed by pacing, verapamil, and lidocaine but not by flunarizine, and 2) the faster VTs are not affected by pacing, lidocaine, and flunarizine, and are only slowed by verapamil. These findings are compatible with in vitro findings of abnormal automaticity, with the slower VTs originating from a higher MDP than the faster VTs.

Detection of benzo[a]pyrene-diol-epoxide-DNA adducts in white blood cells of psoriatic patients treated with coal tar.

An enzyme-linked immunosorbent assay (ELISA) was used to detect BPDE-DNA adducts in white blood cells of 23 psoriatic patients undergoing clinical coal tar therapy. Ten of these patients were reanalyzed 2-5 months after the end of the coal tar treatments. The results show that the mean adduct level during the treatment period was 0.26 +/- 0.16 fmole BPDE/micrograms DNA (7.7 +/- 4.9 adducts/10(8) nucleotides), while 2-5 months later the mean adduct level had decreased significantly (P less than 0.005) to 0.11 +/- 0.08 fmole BPDE/micrograms DNA (3.3 +/- 2.4 adducts/10(8) nucleotides). No relationship could be ascertained between the level of exposure and the amount of BPDE-DNA adducts. In addition, no difference in the level of DNA adducts was found between smoking and non-smoking patients.