Toxicological evaluation of brominated vegetable oil in Sprague Dawley rats.

Brominated vegetable oil (BVO) has been approved by the US Food and Drug Administration on an interim basis as a food additive. Past studies have raised concerns about potential toxicities from consuming BVO. To investigate further these toxicities, we conducted a 90-day dietary exposure study in Sprague Dawley rats and analyzed tissue distribution of the main metabolites. Six-week-old male and female rats were fed diets containing 0 (control), 0.002%, 0.02%, 0.1%, or 0.5% BVO by weight. Statistically significant increases were observed in the serum bromide in the high-dose group of both sexes and in the incidence of thyroid follicular cell hypertrophy in the two highest dose groups of males and the high-dose group of females. An increase in serum TSH was observed in the high-dose group for both sexes, as well as a decrease in serum T4 in the high-dose males. A clear dose-response was observed in di- and tetra-bromostearic acid levels in the heart, liver, and inguinal fat. These data expand upon previous observations in rats and pigs that oral exposure to BVO is associated with increased tissue levels of inorganic and organic bromine, and that the thyroid is a potential target organ of toxicity.

Evaluation of serum and liver toxicokinetics for furan and liver DNA adduct formation in male Fischer 344 rats.

Furan is a food processing contaminant found in many common cooked foods that induces liver toxicity and liver cancer in animal models treated with sufficient doses. The metabolism of furan occurs primarily in the liver where CYP 2E1 produces a highly reactive bis-electrophile, cis-2-butene-1,4-dial (BDA). BDA reacts with nucleophilic groups in amino acids and DNA in vitro to form covalent adducts. Evidence for BDA-nucleoside adduct formation in vivo is limited but important for assessing the carcinogenic hazard of dietary furan. This study used controlled dosing with furan in Fischer 344 rats to measure serum and liver toxicokinetics and the possible formation of BDA-nucleoside adducts in vivo. After gavage exposure, furan concentrations in the liver were consistently higher than those in whole blood (∼6-fold), which is consistent with portal vein delivery of a lipophilic compound into the liver. Formation of BDA-2'-deoxycytidine in furan-treated rat liver DNA was not observed using LC/MS/MS after single doses as high as 9.2 mg/kg bw or repeated dosing for up to 360 days above a consistent background level (1-2 adducts per 10(8) nucleotides). This absence of BDA-nucleoside adduct formation is consistent with the general lack of evidence for genotoxicity of furan in vivo.

Toxicology and carcinogenesis studies of a nondecolorized [corrected] whole leaf extract of Aloe barbadensis Miller (Aloe vera) in F344/N rats and B6C3F1 mice (drinking water study).

BACKGROUND: Extracts from the leaves of the Aloe vera plant (Aloe barbadensis Miller) have long been used as herbal remedies and are also now promoted as a dietary supplement, in liquid tonics, powders or tablets, as a laxative and to prevent a variety of illnesses. We studied the effects of Aloe vera extract on rats and mice to identify potential toxic or cancer-related hazards. METHODS: We gave solutions of nondecolorized extracts of Aloe vera leaves in the drinking water to groups of rats and mice for 2 years. Groups of 48 rats received solutions containing 0.5%, 1% or 1.5% of Aloe vera extract in the drinking water, and groups of mice received solutions containing 1%, 2%, or 3% of Aloe vera extract. Similar groups of animals were given plain drinking water and served as the control groups. At the end of the study tissues from more than 40 sites were examined for every animal. RESULTS: In all groups of rats and mice receiving the Aloe vera extract, the rates of hyperplasia in the large intestine were markedly increased compared to the control animals. There were also increases in hyperplasia in the small intestine in rats receiving the Aloe vera extract, increases in hyperplasia of the stomach in male and female rats and female mice receiving the Aloe vera extract, and increases in hyperplasia of the mesenteric lymph nodes in male and female rats and male mice receiving the Aloe vera extract. In addition, cancers of the large intestine occurred in male and female rats given the Aloe vera extract, though none had been seen in the control groups of rats for this and other studies at this laboratory. CONCLUSIONS: We conclude that nondecolorized Aloe vera caused cancers of the large intestine in male and female rats and also caused hyperplasia of the large intestine, small intestine, stomach, and lymph nodes in male and female rats. Aloe vera extract also caused hyperplasia of the large intestine in male and female mice and hyperplasia of the mesenteric lymph node in male mice and hyperplasia of the stomach in female mice.

The role of UDP-glucuronosyltransferases and drug transporters in breast cancer drug resistance.

One of the major limitations of chemotherapy is that often, over time, tumor cells become either inherently resistant or develop multidrug resistance to the treatment. Another limitation of chemotherapy is toxicity to normal tissues and adverse side effects. The reasons for the failure of some cancers to respond to chemotherapeutic drugs are not clear but have been attributed to alterations in many molecular pathways, which include drug metabolizing enzymes and drug transporter genes. Alterations in the energy-dependent ATP-binding cassette (ABC) transporter genes have been suggested to confer a drug-resistant phenotype by decreasing the intracellular accumulation of chemotherapeutic drugs via efflux mechanisms. In addition, polymorphisms in UDP-glucuronosyltransferases (UGTs) have been reported to correlate with clinical outcome and drug resistance. In this review, we provide an overview of known polymorphisms within UGTs and ABC transporter genes that have been reported to have altered expression and/or activity in breast cancer. Those polymorphic variants that affect the clinical efficacy and confer drug resistance of chemotherapeutic agents, including hormonal therapies, taxanes, anthracyclines, and alkylating agents, in breast cancer.

Oxidative stress related DNA adducts in the liver of female rats fed with sunflower-, rapeseed-, olive- or coconut oil supplemented diets.

Both animal and epidemiological studies support an effect of fatty acid composition in the diet on cancer development, in particular on colon cancer. We investigated the modulating effect of supplementation of the diet of female F344 rats with sunflower-, rapeseed-, olive-, or coconut oil on the formation of the promutagenic, exocyclic DNA adducts in the liver, an organ where major metabolism of fatty acids takes place. 1,N(6)-ethenodeoxyadenosine (etheno-dA), 3,N(4)-ethenodeoxycytidine (etheno-dC) and 1,N(2)-propandodeoxyguanosine from 4-hydroxy-2-nonenal (HNE-dGp) were determined as markers for DNA-damage derived from lipid peroxidation products and markers for oxidative stress. 8-Oxo-deoxyguanosine (8-Oxo-dG) was also measured as direct oxidative stress marker. The body weight of the rats was not influenced by the four diets containing the different vegetable oils during the 4-week feeding period. Highest adduct levels of etheno-dC (430 +/- 181 adducts/10(9) parent bases), HNE-dGp (617 +/- 96 adducts/10(9) parent bases) and 8-Oxo-dG (37,400 +/- 12,200 adducts/10(9) parent bases) were seen in rats on sunflower oil diet (highest linoleic acid content). Highest adducts levels of etheno-dA (133 +/- 113 adducts/10(9) parent bases) were found in coconut oil diet (lowest content of linoleic acid). Weakly positive correlations between linoleic acid content in the four diet groups were only observed for levels of HNE-dGp and 8-Oxo-dG. Neither the diet based on olive oil (which contains mainly oleic acid) nor the diet based on rapeseed oil (containing alpha-linolenic acid) exerted any significant protective effect against oxidative DNA damage. Our results indicate that a high linoleic acid diet may contribute to oxidative stress in the liver of female rats leading to a marginal increase in oxidative DNA-damage.

Cross-cultural adaptation and validation of the NASS outcomes instrument in Spanish patients with low back pain.

OBJECTIVES: The aim of the study was to evaluate validity, reliability, responsiveness and practicality of the NASS-AAOS (North American Spine Society--American Academy of Orthopaedic Surgeons) questionnaire in patients with low back pain. METHODS: The sample included 70 patients with herniated disk, stenosis, chronic low back pain of unknown etiology or acute low back pain. They were assessed twice before treatment (test--retest) and a third measure six months to one year afterwards. RESULTS: The mean time of administration was 24 and 20 min for the test and post-treatment evaluation, respectively. Cronbach's alpha coefficient was between 0.78 and 0.92 on the baseline test and 0.90 or higher on the post-treatment evaluation. The test--retest reproducibility was 0.95 (0.91--0.98) for 'neurological symptoms', 0.82 (0.63--0.91) for 'pain/disability' and 0.63 (0.25--0.82) for 'expectations'. The associations with other measures and clinical criteria were generally moderate to high and in the expected direction. The effect size for 'pain/disability' in combination with 'neurological symptoms' was 2.02 for patients who improved versus an effect of -0.09 in patients who were stable between test and retest; the area under the curve on this joint scale was 0.81 (0.69--0.90). CONCLUSIONS: The instrument is valid, sensitive to clinical changes and reliable for comparisons between groups, but further study is needed for its application in monitoring individual patients.

Analysis of mutations and bone marrow micronuclei in Big Blue rats fed leucomalachite green.

Leucomalachite green (LMG) is the major metabolite of malachite green (MG), a triphenylmethane dye that has been used widely as an antifungal agent in the fish industry. Concern over MG and LMG is due to the potential for consumer exposure, suggestive evidence of tumor promotion in rodent liver, and suspicion of carcinogenicity based on structure-activity relationships. In order to evaluate the risks associated with exposure to LMG, female Big Blue rats were fed up to 543 ppm LMG; groups of these rats were killed after 4, 16, or 32 weeks of exposure and evaluated for genotoxicity. We previously reported that this treatment resulted in a dose-dependent induction of liver DNA adducts, and that the liver lacI mutant frequency (MF) was increased, but only in rats fed 543 ppm LMG for 16 weeks. In the present study, we report the results from lymphocyte Hprt mutant assays and bone marrow micronucleus assays performed on these same rats. In addition, we have determined the types of lacI mutations induced in the rats fed 543 ppm LMG for 16 weeks and the rats fed control diet. No significant increases in the frequency of micronuclei or Hprt mutants were observed for any of the doses or time points assayed. Molecular analysis of 80 liver lacI mutants from rats fed 543 ppm LMG for 16 weeks revealed that 21% (17/80) were clonal in origin and that most (55/63) of the independent mutations were base pair substitutions. The predominant type of mutation was G:C --> A:T transition (31/63) and the majority (68%) of these involved CpG sites. When corrected for clonality, the 16-week lacI mutation frequency (36 +/- 10) x 10(-6) in treated rats was not significantly different from the clonally corrected control frequency (17 +/- 9 x 10(-6); P = 0.06). Furthermore, the lacI mutational spectrum in treated rats was not significantly different from that found for control rats (P = 0.09). Taken together, these data indicate that the DNA adducts produced by LMG in female rats do not result in detectable levels of genotoxicity, and that the increase in lacI MF observed previously in the liver of treated rats may be due to the disproportionate expansion of spontaneous lacI mutations.

Social networks and self-rated health in two French-speaking Canadian community dwelling populations over 65.

The objective was to evaluate the associations between older persons' health status and their social integration and social networks (family, children, friends and community), in two French-speaking, Canadian community dwelling populations aged 65 years and over, using the conceptual framework proposed by Berkman and Thomas. Data were taken from two 1995 surveys conducted in the city of Moncton (n = 1518) and the Montreal neighbourhood of Hochelaga-Maisonneuve (n = 1500). Social engagement (a cumulative index of social activities), networks consisting of friends, family and children and social support were measured using validated scales. Multiple logistic regressions based on structured inclusion of potentially mediating variables were fitted to estimate the associations between health status and social networks. Self-rated health was better for those with a high level of social integration and a strong network of friends in both locations. In addition, in Hochelaga-Maisonneuve family and children networks were positively associated with good health, though the effect of friend networks was attenuated in the presence of disability, good social support from children was associated with good health. Age, sex and education were included as antecedent variables; smoking, alcohol consumption, exercise, locus of control and depressive symptoms were considered intermediary variables between social networks and health. In conclusion, social networks, integration and support demonstrated unique positive associations with health. The nature of these associations may vary between populations and cultures.

Mutagenicity and carcinogenicity in relation to DNA adduct formation in rats fed leucomalachite green.

Leucomalachite green is a persistent and prevalent metabolite of malachite green, a triphenylmethane dye that has been used widely as an antifungal agent in the fish industry. Concern over the use of malachite green is due to the potential for consumer exposure, evidence suggestive of tumor promotion in rodent liver, and suspicion of carcinogenicity based on structure-activity relationships. Our previous study indicated that feeding rodents malachite or leucomalachite green resulted in a dose-related increase in liver DNA adducts, and that, in general, exposure to leucomalachite green caused an increase in the number and severity of changes greater than was observed following exposure to malachite green. To characterize better the genotoxicity of leucomalachite green, female Big Blue rats were fed leucomalachite green at doses of 0, 9, 27, 91, 272, or 543 ppm for up to 32 weeks. The livers were analyzed for lacI mutations at 4, 16, and 32 weeks and DNA adducts at 4 weeks. Using a 32P-postlabeling assay, we observed a dose-related DNA adduct in the livers of rats fed 91, 272, and 543 ppm leucomalachite green. A approximately 3-fold increase in lacI mutant frequency was found in the livers of rats fed 543 ppm leucomalachite green for 16 weeks, but significant increases in mutant frequencies were not found for any of the other doses or time points assayed. We also conducted 2-year tumorigenesis bioassays in female and male F344 rats using 0, 91, 272, and 543 ppm leucomalachite green. Preliminary results indicate an increasing dose trend in lung adenomas in male rats treated with leucomalachite green, but no increase in the incidence of liver tumors in either sex of rat. These results suggest that the DNA adduct formed in the livers of rats fed leucomalachite green has little mutagenic or carcinogenic consequence.

Correlates of wellbeing of spousal and children carers of disabled people over 65 in Spain.

BACKGROUND: The aim of this study was to evaluate the associations between carer's wellbeing and stressors and to assess if these associations are different for spousal and children carers of disabled elderly. METHODS: Information was collected by home interviews of a population sample of carers (N = 195), who were providing assistance in activities of daily living to a community-dwelling population over 65. Associations between indicators of wellbeing (number of depressive symptoms, number of physical symptoms, self-perceived health and life satisfaction) and caring stressors were examined, controlling for carer's socio-economic characteristics and health status. Hierarchical logistic regressions were used to fit the data. Religion and social support were included as resources and spousal and children differential associations were tested. RESULTS: The four indicators of wellbeing are moderately correlated, indicating a common underlying concept. Spousal carers have lower socio-economic status, poorer health and lower levels of wellbeing than children carers. However, children carers bear a significantly greater burden. In the multivariate analysis of the associations between wellbeing and stressors, the similarities between spouse and adult child carers are more striking than the differences. Emotional support was consistently associated with higher levels of wellbeing while the associations of religiosity and instrumental support with wellbeing did not reach statistical significance. CONCLUSION: As formal care services are being developed in Spain, their ability to work in a supportive way with family networks should be taken into account. Research should be carried out on patterns of formal care interventions that use the resources in the natural support network of the family.

Analysis of DNA adducts from chemical carcinogens and lipid peroxidation using liquid chromatography and electrospray mass spectrometry.

The identification and dosimetry of DNA adducts are cornerstones of research on cancer etiology in experimental animals and humans. DNA adducts can result from exposure to exogenous chemical carcinogens or through reactions with endogenous by-products of oxidative metabolism. An important research need is high throughput methodology for quantification of any and all adducts that are present at trace amounts in DNA derived from target tissues of animals and humans. This review describes some recent progress made through applications of liquid chromatography coupled with mass spectrometry to structural characterization of unknown DNA adducts and highly sensitive quantitative analysis of target adducts.

Support from children, living arrangements, self-rated health and depressive symptoms of older people in Spain.

OBJECTIVE: To assess the association between emotional and instrumental support from children and living arrangements with the physical and mental health of older people in Spain. METHODS: A face-to-face home interview was carried out with 1284 community-dwelling people over 65 (response rate = 83%) randomly sampled according to an age- and sex-stratified sampling scheme in 1993 at Leganés (Spain). Close to 93% of the participants had children and 45% of them coresided with them. Depressive symptoms were assessed by the CES-D (Center for Epidemiologic study depression scale) and self-rated health (SRH) by a single-item question. Emotional support was measured with a six-item scale on affection and reciprocity. Instrumental support was assessed by help received from children in 17 activities of daily living. Four living arrangements were considered: Living with spouse only, living with a spouse and children, widower living alone, and widower living with children. RESULTS: Multivariate analysis controlling for age, gender, education and functional status showed that low emotional support and reception of instrumental aid were significantly associated with poor SRH. Being a widower and sharing living arrangements with children was associated with good SRH. Living arrangements modify some of the associations of support of children with SRH. Depressive symptoms were associated with low emotional support, reception of instrumental help and being a widower who did not share living arrangements with children. For widowers who do not cohabit with children, reception of instrumental aid is associated with low depressive symptomatology. DISCUSSION: Emotional support from children seems to play an important role in maintaining the physical and mental health of elderly people in Spain. Instrumental support is widely available. Coresidence with children is very common and it is associated with good self-perceived health and low prevalence of depressive symptoms in a culture where family interdependence is highly valued. Families should be protected and encouraged to continue care-giving through a variety of community services and respite care, adapted to their needs and preferences. Research should be undertaken to find more efficient ways to help family caregivers in the Mediterranean context.

The effect of medical work groups on hospital resource use.

This study tests the ability of medical work groups to overcome coordination problems related to group decision-making in allocating clinical resources to inpatients. The study was conducted over a 32-month period in two medium-sized acute-care hospitals located in Montreal, Quebec, Canada. The data were collected by hand from the medical charts of 10,456 patients in the surgical and medical departments. The Linear Structural Relations (LISREL) approach was employed to address the work-group issue using a task contingent model of work-group organization. In this model, the nature of the task is fundamental because its level of complexity determines both the organization of the work group and the use of resources. Medical work-group mechanisms should be efficient to the extent that resource utilization is explained solely by task characteristics rather than by work-group structure. In this study, the following two major organizational concepts were used as factors to explain resource use: task characteristics and work-group characteristics. Our analysis confirmed the main points of the task contingency theory as applied to the field of medicine. First, the results confirm that resource utilization is explained mainly by task complexity. Second, they confirm that medical work groups modulate their structures on the basis of task characteristics and do not explain resource use. The results also reveal a more complex model in which, for instance, the concepts of medical task and medical professional work are not easy to separate. The results highlight the interest in conceptualizing and analysing medical practice in work groups. It raises important issues that have seldom been taken into account in the study of medical practice variations, which has tended to focus on attending physicians.

DNA adduct formation and mutant induction in Sprague-Dawley rats treated with tamoxifen and its derivatives.

The non-steroidal anti-estrogen tamoxifen is used as an adjunct chemotherapeutic agent for the treatment of all stages of breast cancer and more recently as a chemoprotective agent in women with elevated risk of developing breast cancer. While beneficial for the treatment of breast cancer, tamoxifen increases the risk of endometrial cancer. In addition, it has been shown to induce liver and endometrial tumors in rats. Tamoxifen is genotoxic in rat liver, as indicated by the formation of DNA adducts, through a metabolic pathway involving the alpha-hydroxylation of tamoxifen and N-desmethyltamoxifen. Since the contribution of these alpha-hydroxy metabolites of tamoxifen to the induction of endometrial tumors is presently unknown, we compared the extent of DNA adduct formation in liver and selected non-hepatic tissues of female Sprague-Dawley rats treated by gavage with tamoxifen, alpha-hydroxytamoxifen, N-desmethyltamoxifen, alpha-hydroxy-N-desmethyltamoxifen and N,N-didesmethyltamoxifen, or intraperitoneal injection with tamoxifen, alpha-hydroxytamoxifen, 3-hydroxytamoxifen and 4-hydroxytamoxifen. In addition, spleen lymphocytes from rats treated by gavage with tamoxifen or alpha-hydroxytamoxifen were assayed for the induction of mutants in the hypoxanthine phosphoribosyl transferase (Hprt) gene. The relative levels of binding in rats treated by gavage were alpha-hydroxytamoxifen > tamoxifen approximately N-desmethyltamoxifen approximately alpha-hydroxy-N-desmethyltamoxifen > N,N-didesmethyltamoxifen. In rats dosed intraperitoneally, the relative order of binding was alpha-hydroxytamoxifen > tamoxifen > 3-hydroxytamoxifen approximately 4-hydroxytamoxifen. None of the compounds resulted in an increase in DNA adducts in uterus, spleen, thymus or bone marrow DNA from rats treated by gavage or in uterus DNA from rats injected intraperitoneally. Neither tamoxifen nor alpha-hydroxytamoxifen increased the Hprt mutant frequency in spleen T-lymphocytes. These results confirm previous observations that tamoxifen is activated to a genotoxic agent in rat liver through alpha-hydroxylation, and also suggest that endometrial tumors in rats do not arise from the formation of tamoxifen-DNA adducts.

Loss to follow-up in a longitudinal study on aging in Spain.

The objectives of this study were to describe follow-up dynamics in a longitudinal study on aging conducted in Spain between 1993 and 1997, and to identify the demographic, behavioral and health characteristics of persons who would later refuse to continue participating, move out of the area or be hard to locate (i.e., become lost to follow-up subjects). Data from the 1993 baseline survey were used to predict the probabilities of being lost to follow-up in the 1995 and 1997 waves. Structural multiple logistic regressions were fitted and mean probabilities were estimated to identify patterns of loss to follow-up. After 4 years, 52% of baseline participants remained in the study, 24% had died, 17% refused to continue participating, and 8.7% were impossible to locate. In the multivariate analysis, advanced age and living alone were independent predictors of loss to follow-up, and none of the health status variables remained significant. However, participation status in previous waves and the number of nonresponse items were strong independent predictors of further non-participation. Our results suggest that an attitude against participation in surveys may be an independent predictor of losses to follow-up and efforts should be made to retain this subgroup of the population in the study. Further research on reasons why people are unwilling to participate in surveys and strategies to retain people in longitudinal studies is needed.

Comparison of hprt and lacI mutant frequency with DNA adduct formation in N-hydroxy-2-acetylaminofluorene-treated Big Blue rats.

N-Hydroxy-2-acetylaminofluorene (N-OH-AAF) is the proximate carcinogenic metabolite of the powerful rat liver carcinogen 2-acetylaminofluorene. In this study, transgenic Big Blue(R) rats were used to examine the relationship between in vivo mutagenicity and DNA adduct formation by N-OH-AAF in the target liver compared with that in nontarget tissues. Male rats were given one, two, or four doses of 25 mg N-OH-AAF/kg body weight by i.p. injection at 4-day intervals, and groups of treated and control rats were euthanized up to 10 weeks after beginning the dosing. Mutant frequencies were measured in the spleen lymphocyte hprt gene, and lacI mutant frequencies were determined in the liver and spleen lymphocytes. At 6 weeks after beginning the dosing, the hprt mutant frequency in spleen lymphocytes from the four-dose group was 16.5 x 10(-6) compared with 3.2 x 10(-6) in control animals. Also at 6 weeks, rats given one, two, or four doses of N-OH-AAF had lacI mutant frequencies in the liver of 97.6, 155.6, and 406.8 x 10(-6), respectively, compared with a control frequency of 25.7 x 10(-6); rats given four doses had lacI mutant frequencies in spleen lymphocytes of 55.8 x 10(-6) compared with a control frequency of 20.4 x 10(-6). Additional rats were evaluated for DNA adduct formation in the liver, spleen lymphocytes, and bone marrow by (32)P-postlabeling. Adduct analysis was conducted 1 day after one, two, and four treatments with N-OH-AAF, 5 days after one treatment, and 9 days after two treatments. N-(Deoxyguanosin-8-yl)-2-aminofluorene was the major DNA adduct identified in all the tissues examined. Adduct concentrations increased with total dose to maximum values in samples taken 1 day after two doses, and remained essentially the same after four doses. In samples taken after four doses, adduct levels were 103, 28, and 7 fmol/microg of DNA in liver, spleen lymphocytes, and bone marrow, respectively. The results indicate that the extent of both DNA adduct formation and mutant induction correlates with the organ specificity for N-OH-AAF carcinogenesis in the rat. Environ. Mol. Mutagen. 37:195-202, 2001. Published 2001 Wiley-Liss, Inc.

Synthesis, characterization, and comparative 32P-postlabeling efficiencies of 2,6-dimethylaniline-DNA adducts.

2,6-Dimethylaniline (2,6-diMeA) is a ubiquitous environmental pollutant that is used in industry as a synthetic intermediate. It is also found in tobacco smoke and as a major metabolite of lidocaine. Although the potential carcinogenicity of 2,6-diMeA in humans is presently uncertain, this aromatic amine has been classified as a rodent carcinogen. In addition, it is known to form hemoglobin adducts in humans, which indicates a profile of metabolic activation similar to that of typical arylamine carcinogens. Like other aromatic amines, 2,6-diMeA has been shown to yield N-(deoxyguanosin-8-yl)-2,6-dimethylaniline (dG-C8-2,6-diMeA) as a major DNA adduct in vitro. In this study, we show that 2,6-diMeA yields an unusual pattern of DNA adducts. In addition to dG-C8-2,6-diMeA, we have isolated two new adducts, 4-(deoxyguanosin-N(2)-yl)-2,6-dimethylaniline (dG-N(2)-2,6-diMeA) and 4-(deoxyguanosin-O(6)-yl)-2,6-dimethylaniline (dG-O(6)-2,6-diMeA), from the reaction of N-acetoxy-2,6-dimethylaniline with deoxyguanosine. A similar reaction conducted with deoxyadenosine yielded 4-(deoxyadenosin-N(6)-yl)-2,6-dimethylaniline (dA-N(6)-2,6-diMeA). All four adducts were detected in DNA reacted with N-acetoxy-2,6-dimethylaniline, with the relative yields being 46% for dA-N(6)-2,6-diMeA, 22% for dG-N(2)-2,6-diMeA, 20% for dG-O(6)-2,6-diMeA, and 12% for dG-C8-2,6-diMeA. This product profile contrasts markedly with the usual pattern of adducts obtained with aromatic amines, where C8-substituted deoxyguanosine products typically predominate. We further analyzed the kinetics of the T(4) polynucleotide kinase (PNK)-catalyzed phosphorylation of the C8 and N(2) deoxyguanosine 3'-phosphate adducts from 2,6-diMeA. The kinetic parameters obtained with these two structurally different adducts are compared to those determined with the parent nucleotide (dG3'p), and with (+/-)-anti-10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene 3'-phosphate, the major adduct derived from the environmental pollutant benzo[a]pyrene. The results indicate that all the adducts were labeled with lower efficiencies than dG3'p, stressing the likely underestimation of adduct levels in typical 32P-postlabeling protocols. Nonetheless, the N(2) adducts derived from 2,6-diMeA and benzo[a]pyrene were both labeled with higher efficiencies than the C8 adduct derived from 2,6-diMeA, with the benzo[a]pyrene adduct being the best substrate for PNK. Thus, the data suggest that N(2) adducts from dG3'p are intrinsically better substrates than their C8 analogues for PNK, and that bulkier aromatic fragments may favor the enzyme-substrate interaction during the labeling step.

Quantitative analysis of etheno-2'-deoxycytidine DNA adducts using on-line immunoaffinity chromatography coupled with LC/ES-MS/MS detection.

Etheno DNA adducts, including 3,N4-etheno-2'-deoxycytidine (etheno-dC), are promutagenic lesions present in normal animal and human tissues. These DNA adducts are believed to be important in the etiology of cancer. Existing methods for quantifying etheno-dC use 32p. postlabeling. Although highly sensitive, postlabeling requires the use of an energetic radioisotope and considerable time and effort. The new methodology reported here permits automated quantification of trace levels of etheno-dC in crude DNA hydrolysates on the order of 5 adducts in 10(8) normal nucleotides from 100-microg samples of DNA. This was accomplished by using on-line immunoaffinity chromatography, a reverse-phase LC separation on graphitized carbon, tandem mass spectrometric detection, and an isotopically labeled internal standard. The automated procedures permitted analysis of 4 DNA hydrolysates/hr. The sensitivity using immunoaffinity cleanup was approximately 100-fold greater than that observed when using a silica-based trapping system. The validated method was applied to the analysis of etheno-dC in commercial calf thymus DNA, untreated mouse liver, and untreated rat liver DNA. The demonstrated level of performance suggests future applicability of this method in studies of cancer in humans and experimental animals.

Quantification of etheno-DNA adducts using liquid chromatography, on-line sample processing, and electrospray tandem mass spectrometry.

Etheno-DNA adducts are promutagenic lesions present in normal animal and human tissues that are believed to be important in the etiology of cancer related to diet and lifestyle. A method has been developed for the quantification of trace levels of etheno-DNA adducts using on-line sample preparation coupled with liquid chromatography and electrospray tandem mass spectrometry. The use of automated solid-phase extraction and stable labeled internal standards permitted the robust determination of ethenodeoxyadenosine contained in crude DNA hydrolysates from untreated rodent and human tissues at levels on the order of one adduct in 10(8) normal nucleotides from 100 microg of DNA. Inherent analyte response and matrix interference made sensitivity for simultaneous determination of ethenodeoxycytidine approximately 5-fold lower. The method was applied to the analysis of liver DNA from untreated and urethane-treated B6C3F1 mice, untreated rat liver, human placenta, and several commercial DNA preparations. Some sources of potential artifactual formation of etheno-DNA adducts were investigated.

Women, drugs and HIV/AIDS: results of a multicentre European study.

BACKGROUND: In the light of rising human immunodeficiency virus (HIV) incidence rates amongst women in Western Europe, a multicentred, cross-sectional study was undertaken to explore the multitude of possible factors associated with HIV in a population of female injecting drug users (IDU). METHODS: Face-to-face interviews were conducted with 1198 female IDU recruited from a variety of settings in Paris, Madrid, Rome, London and Berlin. Their HIV status was determined from antibody testing of blood or saliva samples or from written confirmation of HIV test results from a physician. A hierarchical logistic regression model was used to identify direct and indirect associations between socioeconomic factors, marginalization and risk behaviour with HIV prevalence. RESULTS: The HIV prevalence in the sample of female IDU was 27.8% (range: 1.4% in London and 52.6% in Madrid). Factors independently associated with HIV prevalence in the regression analysis included: age >25 years (OR = 2.0-2.9), left full-time education before age 14 (OR = 2.4), no fixed address (OR = 2.2), previous imprisonment (OR = 1.4), commercial sex (OR = 1.3), having a regular HIV positive sexual partner (OR = 6.6), ever shared needles (OR = 1.5) and any sexually transmitted disease (STD) infection in the last year (OR = 1.7). CONCLUSIONS: The sexual behaviour and partners of female IDU in Western Europe are as important a component in explaining the HIV epidemic in this population as other risk factors, including high-risk drug taking behaviour. Homeless IDU women may be an important residual risk group warranting future preventive interventions and women with a history of STD should be a particular target for health education. Differences in HIV prevalence across cities are very large and may be related to differences in harm reduction policies.