Does the gene matter? Genotype-phenotype and genotype-outcome associations in congenital melanocytic naevi.

BACKGROUND: Genotype-phenotype studies can identify subgroups of patients with specific clinical features or differing outcomes, which can help shape management. OBJECTIVES: To characterize the frequency of different causative genotypes in congenital melanocytic naevi (CMN), and to investigate genotype-phenotype and genotype-outcome associations. METHODS: We conducted a large cohort study in which we undertook MC1R genotyping from blood, and high-sensitivity genotyping of NRAS and BRAF hotspots in 156 naevus biopsies from 134 patients with CMN [male 40%; multiple CMN 76%; projected adult size (PAS) > 20 cm, 59%]. RESULTS: Mosaic NRAS mutations were detected in 68%, mutually exclusive with BRAF mutations in 7%, with double wild-type in 25%. Two separate naevi were sequenced in five of seven patients with BRAF mutations, confirming clonality. Five of seven patients with BRAF mutations had a dramatic multinodular phenotype, with characteristic histology distinct from classical proliferative nodules. NRAS mutation was the commonest in all sizes of CMN, but was particularly common in naevi with PAS > 60 cm, implying more tolerance to that mutation early in embryogenesis. Facial features were less common in double wild-type patients. Importantly, the incidence of congenital neurological disease, and apparently of melanoma, was not altered by genotype; no cases of melanoma were seen in BRAF-mutant multiple CMN, however, this genotype is rare. CONCLUSIONS: CMN of all sizes are most commonly caused by mutations in NRAS. BRAF is confirmed as a much rarer cause of multiple CMN, and appears to be commonly associated with a multinodular phenotype. Genotype in this cohort was not associated with differences in incidence of neurological disease in childhood. However, genotyping should be undertaken in suspected melanoma, for guidance of treatment. What's already known about this topic? Multiple congenital melanocytic naevi (CMN) have been shown to be caused by NRAS mosaic mutations in 70-80% of cases, by BRAF mosaicism in one case report and by inference in some previous cases. There has been debate about genotypic association with different sizes of CMN, and no data on genotype-outcome. What does this study add? NRAS mosaicism was found in 68%, BRAF in 7% and double wild-type in 25% of cases of CMN. NRAS was the commonest mutation in all sizes of CMN, but was nearly universal in projected adult size > 60 cm. BRAF is often associated with a distinct multinodular clinical/histological phenotype. Adverse outcomes did not differ between genotypes on current numbers.

Citizens advice in primary care: a qualitative study of the views and experiences of service users and staff.

OBJECTIVES: To examine the views and experiences of staff and users of Citizens Advice Bureau (CAB) services located in general practice, and to identify key factors perceived as contributing to the intervention's effectiveness. STUDY DESIGN: A qualitative study in an urban and rural primary care setting in the UK. METHODS: Semi-structured, face-to-face interviews (n = 22) with primary care and practice staff, CAB advisors and 12 service users. RESULTS: Key positive service features reported by all groups were: the confidential, non-stigmatizing and familiar environment of a general practitioner's (GP) surgery; the ability to make appointments and experienced advisor availability and continuity. Outcomes for service users were described as financial gain, managed debt, and beneficial social and mental health impacts. Perceived staff benefits were appropriate referral and better use of GP consultation time. CONCLUSION: Welfare advice in primary care has financial benefits and was perceived by participants to offer health and other benefits to patients and staff. However, while perceptions of gain from the intervention were evident, demonstration of measurable health improvement and well-being presents challenges. Further empirical work is needed in order to explore these complex cause-effect links and the cost-effectiveness of the intervention.

VEGF blockade decreases the tumor uptake of systemic oncolytic herpes virus but enhances therapeutic efficacy when given after virotherapy.

Effective therapies for metastatic sarcomas remain elusive. Oncolytic viruses have shown promise as anticancer agents, but their access to metastatic sites following systemic delivery is low. As systemic delivery of small-molecule chemotherapy is enhanced by previous treatment with antiangiogenic agents because of changes in intravascular-to-tumor interstitial pressure, we sought to determine whether antiangiogenic pretreatment increases the antitumor efficacy of systemic virotherapy by increasing virus uptake into tumor. Virus biodistribution and antitumor effects were monitored in tumor-bearing mice given antihuman vascular endothelial growth factor (VEGF) or antimouse VEGFR2 before or after an intravenous (i.v.) injection of virus. Without pretreatment, the average virus titers in the tumor samples amplified 1700-fold over 48 h but were undetectable in other organs. After antiangiogenic treatment, average virus titers in the tumor samples were unchanged or in some cases decreased up to 100-fold. Thus, antiangiogenic pretreatment failed to improve the tumor uptake of systemic oncolytic herpes simplex virus (oHSV), in contrast to previously reported enhanced uptake of small molecules. Superior tumor control because of the combined effects of virus and anti-VEGF was seen most dramatically when anti-VEGF was given after virus. Our data suggest that i.v. oHSV can treat distant sites of disease and can be enhanced by antiangiogenic therapy, but only when given in the proper sequence.

Holding personal information in a disease-specific register: the perspectives of people with multiple sclerosis and professionals on consent and access.

OBJECTIVE: To determine the views of people with multiple sclerosis (MS) and professionals in relation to confidentiality, consent and access to data within a proposed MS register in the UK. DESIGN: Qualitative study using focus groups (10) and interviews (13). SETTING: England and Northern Ireland. PARTICIPANTS: 68 people with MS, neurologists, MS nurses, health services management professionals, researchers, representatives from pharmaceutical companies and social care professionals. RESULTS: People with MS expressed open and altruistic views towards the use of their personal information to facilitate service provision and research, placing trust in responsible guardianship and legitimate use of their information. Participant's proposed that people with MS should be able to select their individual level of involvement in a register using levels of consent. It was agreed that access to the register should be governed by a guardianship committee composed of a range of stakeholders. People with MS did not wish their details to be used by marketing agencies and did not consider this a legitimate use of their data. Whilst participants were positive of the role a register could play in promoting research, participants felt that access to data by pharmaceutical industries should be administered by the guardianship committee. People with MS are concerned should their employers be able to access their personal information. Professionals were more cautious than people with MS in their approach to the use of patient personal data within a register. CONCLUSIONS: Whilst all stakeholders were positive of the benefits of an MS register, development of such a resource must incorporate robust data security and guardianship measures in order to ensure that, whilst opportunities are maximised, risks to the privacy of individuals and legal challenges to professionals are avoided.

Molecular analysis of human cancer cells infected by an oncolytic HSV-1 reveals multiple upregulated cellular genes and a role for SOCS1 in virus replication.

Oncolytic herpes simplex viruses (oHSVs) are promising anticancer therapeutics. We sought to characterize the functional genomic response of human cancer cells to oHSV infection using G207, an oHSV previously evaluated in a phase I trial. Five human malignant peripheral nerve sheath tumor cell lines, with differing sensitivity to oHSV, were infected with G207 for 6 h. Functional genomic analysis of virus-infected cells demonstrated large clusters of downregulated cellular mRNAs and smaller clusters of those upregulated, including 21 genes commonly upregulated in all five lines. Of these, 7 are known to be HSV-1 induced and 14 represent novel virus-regulated genes. Gene ontology analysis revealed that a majority of G207-upregulated genes are involved in Janus kinase/signal transducer and activator of transcription signaling, transcriptional regulation, nucleic acid metabolism, protein synthesis and apoptosis. Ingenuity networks highlighted nodes for AP-1 subunits and interferon signaling via STAT1, suppressor of cytokine signaling-1 (SOCS1), SOCS3 and RANTES. As biological confirmation, we found that virus-mediated upregulation of SOCS1 correlated with sensitivity to G207 and that depletion of SOCS1 impaired virus replication by >10-fold. Further characterization of roles provided by oHSV-induced cellular genes during virus replication may be utilized to predict oncolytic efficacy and to provide rational strategies for designing next-generation oncolytic viruses.

Development of transformation vectors based upon a modified plant alpha-tubulin gene as the selectable marker.

A plant transformation and selection system has been developed utilizing a modified tubulin gene as a selectable marker. The vector constructs carrying a mutant alpha-tubulin gene from goosegrass conferring resistance to dinitroaniline herbicides were created for transformation of monocotyledonous and dicotyledonous plants. These constructs contained beta- and/or mutant alpha-tubulin genes driven either by ubiquitin or CaMV 35S promoter. The constructs were used for biolistic transformation of finger millet and soybean or for Agrobacterium-mediated transformation of flax and tobacco. Trifluralin, the main representative of dinitroaniline herbicides, was used as a selective agent in experiments to select transgenic cells, tissues and plantlets. Selective concentrations of trifluralin estimated for each species were as follows: 10 microM for Eleusine coracana, Glycine max, Nicotiana plumbaginifolia and Nicotiana sylvestris; 3 microM for Linum usitatissimum. PCR and Southern blotting analyses of transformed lines with a specific probe to nptII, alpha-tubulin or beta-tubulin genes were performed to confirm the transgenic nature of regenerated plants. Band specific for the mutant alpha-tubulin gene was identified in transformed plant lines. Results confirmed the stable integration of the mutant tubulin gene into the plant genomes. The present study clearly demonstrates the use of a plant mutant tubulin as a selective gene for plant transformation.

Access to dental services for people with a physical disability: a survey of general dental practitioners in Leicestershire, UK.

OBJECTIVE: To investigate the availability of facilities, including parking, accessibility and toilet amenities, for physically disabled people at dental practices in Leicestershire, and views relating to the provision of treatment, as reported by general dental practitioners. BASIC RESEARCH DESIGN: A cross-sectional postal questionnaire-based study. SETTING: General Dental Service practices in Leicestershire, United Kingdom. PARTICIPANTS: Questionnaires were sent to all General Dental Service practices (n=123) within Leicestershire. MAIN OUTCOME MEASURES: Facilities for physically disabled people as reported by general dental practitioners and views of practitioners in relation to provision of treatment. RESULTS: The response rate from general dental practices was 80%. The views of 120 (42%) of the 284 dentists approached relating to the provision of treatment to people with a physical disability were recorded. Although up to 77% of the dental practices were considered by practitioners to be accessible to someone using a wheelchair, only 7% also had suitable parking and toilet facilities. The majority of responding dentists treated patients with a physical disability, but 76% of practitioners found it difficult to provide treatment to this group. Concerns regarding the financial cost of providing treatment were raised. There is evidence that conditions are less than optimal in general practice settings for patients with a physical disability receiving treatment. Only nine of the 123 practices in Leicestershire had appropriate parking, access and toilet facilities for physically disabled people. CONCLUSION(S): Facilities for physically disabled people at general practices in Leicestershire are limited. If inequalities in dental health among the physically disabled are to be successfully reduced, steps must be taken to make practices more easily accessible with suitable facilities, and to increase awareness of services offered by appropriate dental practices.

Perspectives of dentists in primary care ahead of the 'new ways of working'.

OBJECTIVE: To determine the future intentions and motivations of general dental practitioners (GDPs) relating to NHS dental practice in South Yorkshire. DESIGN: Focus group discussions. SETTING: General dental practices providing NHS care within South Yorkshire, United Kingdom. SUBJECTS (MATERIALS) AND METHODS: Twenty-nine dental practitioners were purposively sampled and invited to take part in a series of focus groups. Focus groups were transcribed and data analysed to identify themes and concepts. MAIN OUTCOME MEASURES: Themes and concepts relating to the current and future provision of dentistry and the proposed 'new ways of working' of the new dental contract. RESULTS: The data fell into three broad categories: the organisational structures of dentistry; the future of dentistry; and the CDS. This paper focuses largely on the second category, the future of NHS dentistry. The first category related to the organisational structures of dentistry, and encompassed perceptions that dentistry was not a high priority for the Government and that current changes were politically motivated and to be implemented by PCTs with a lack of capacity for the management of such wide-reaching changes. The second category covered the future of NHS dentistry. For some, NHS dentistry was in a precarious and uncertain position, coupled with a lack of clarity and information on the 'new ways of working' and exacerbated by problems in the recruitment and retention of future dental practitioners. The last category dealt with views in connection with the CDS. CONCLUSION(S): In this 'snapshot in time' there was considerable uncertainty and instability within the general dental service against a backdrop of major organisational change. There was a need for information, guidance, openness and communication between the Government, PCTs and GDPs surrounding the implementation of the new contract.

Verifiable CPD paper: factors that influence the dental attendance pattern and maintenance of oral health for people with multiple sclerosis.

OBJECTIVE: To determine the impact of multiple sclerosis (MS) on patient attendance at dental practices and maintenance of oral health. DESIGN: A cross-sectional postal questionnaire-based study. SETTING: Leicestershire, United Kingdom. SUBJECTS AND METHODS: People with MS in Leicestershire identified from local health authority records (n = 476). MAIN OUTCOME MEASURES: Number registered at dental practice, frequency of attendance, issues and perspectives relating to attendance and maintenance of oral health. RESULTS: A response rate of 61% (n = 289) was obtained. When compared to the general population, a higher number of people with MS were registered with a dentist (49%:88%) and displayed more frequent practice attendance (71%:81%) in the past year. People with MS reported difficulties in attending a dentist and maintaining oral health, which were exacerbated by deterioration in general health. Problems relating to reduced personal mobility had the greatest impact on attendance. CONCLUSIONS: MS has a negative impact on perceived patient attendance and maintenance of oral health. Patients with a progressive disability could benefit greatly from the provision of preventive oral health care. The importance of seeking care earlier rather than later needs to be emphasised to both professionals and patients alike. Further efforts are required to increase awareness of the importance of oral health to the quality of life of people with MS and ensure that individuals with physical disabilities receive the same access to dental services as the able-bodied.

Comparative in vitro metabolism of benzo[a]pyrene by recombinant zebrafish CYP1A and liver microsomes from beta-naphthoflavone-treated rainbow trout.

The zebrafish (Danio rerio) is a sensitive non-mammalian model used for studying polycyclic aromatic hydrocarbon (PAH)-induced chemical carcinogenesis. The susceptibility of zebrafish to PAH-induced carcinogenesis may be related to the ability of the zebrafish P450s to bioactivate these procarcinogens. As a part of our overall effort to identify the various P450 enzymes that are involved in the activation and detoxification of PAHs in zebrafish, therefore, we have examined the ability of recombinant zebrafish CYP1A (zCYP1A) expressed in yeast to metabolize BaP in vitro. Comparison studies also were conducted with liver microsomes from beta-naphthoflavone (BNF)-treated rainbow trout (Oncorhynchus mykiss). Results demonstrated that the trout liver microsomes were almost twice as active as zCYP1A in oxidizing BaP, with Vmax values of 1.7 and 0.94 nmol/min/nmol P450 for trout and zebrafish preparations, respectively. Like trout CYP1A1, cDNA-expressed zCYP1A was found to oxidize BaP to phenols, quinones and diols (BaP-7,8-diol and BaP-9,10-diol) in the presence of exogenous human microsomal epoxide hydrolase (hEH). BaP-7,8-diol is the precursor of the ultimate carcinogen, BaP-7,8-diol-9,10-epoxide (BaPDE). The ability of zCYP1A to bioactivate BaP was confirmed by the formation of DNA adducts when calf thymus DNA was added to the incubation mixture. BaP-DNA binding was enhanced by the addition of hEH to the incubation mixture. HPLC analysis of the [33P]-postlabeled DNA adducts showed the formation of at least four adducts mediated by both zCYP1A and trout liver microsomes, and one of these adducts co-migrated with BaPDE-dG in HPLC analysis. The addition of hEH to the incubation mixture decreased the formation of BaPDE-dG by zCYP1A and by trout liver microsomes while increasing the formation of an unidentified DNA adduct in the case of zCYP1A. zCYP1A also mediated the binding of BaP to protein, providing further evidence that this enzyme is capable of oxidizing BaP to reactive metabolites that bind to macromolecules. It thus appears that zCYP1A may play an important role in BaP-induced carcinogenesis in the zebrafish model by catalyzing the sequential formation of the ultimate diol epoxide carcinogenic metabolite of BaP.

The ribosomal small-subunit protein S28 gene from Helianthus annuus (Asteraceae) is down-regulated in response to drought, high salinity, and abscisic acid.

A partial cDNA for the ribosomal S28 gene from sunflower was initially cloned and identified to be down-regulated by high salinity, using differential display reverse transcriptase-polymerase chain reaction (PCR). Using this sequence, a 502-base pair (bp) full-length cDNA was cloned by rapid amplification of cDNA ends. This cDNA (designated Ha-RPS28) encodes a protein component of the small subunit of cytoplasmic ribosomes. The predicted 65 amino acid residue sequence of Ha-RPS28, with an estimated molecular mass of 7.5 kD, has 92, 89, and 86% identity with the S28 ribosomal proteins from peach, maize, and Arabidopsis, respectively. Ha-RPS28 was expressed in all organs examined, and the highest level was detected in fully expanded leaves. Furthermore, expression of Ha-RPS28 was down-regulated in both seedling roots and shoots in response to drought, high salinity, or abscisic acid.

Effect of a complex environmental mixture from coal tar containing polycyclic aromatic hydrocarbons (PAH) on the tumor initiation, PAH-DNA binding and metabolic activation of carcinogenic PAH in mouse epidermis.

Human exposure to polycyclic aromatic hydrocarbons (PAH) occurs through complex mixtures such as coal tar. The effect of complex PAH mixtures on the activation of carcinogenic PAH to DNA-binding derivatives and carcinogenesis were investigated in mice treated topically with NIST (National Institute of Standards and Technology) Standard Reference Material 1597 (SRM), a complex mixture of PAH extracted from coal tar, and either additional benzo[a]pyrene (B[a]P) or dibenzo[a,l]pyrene (DB[a,l]P). In an initiation-promotion study using 12-O-tetradecanoylphorbol-13-acetate as the promoter for 25 weeks, the SRM and B[a]P co-treated mice had a similar incidence of papillomas per mouse compared with the group exposed to B[a]P alone as the initiator. PAH-DNA adduct analysis of epidermal DNA by 33P-post-labeling and reversed-phase high-performance liquid chromatography found the SRM co-treatment led to a significant decrease in the total level of DNA adducts and B[a]P-DNA adducts to less than that observed in mice treated with B[a]P alone at 6, 12 and 72 h exposure. After 24 and 48 h exposure, there was no significant difference in the levels of adducts between these groups. In the DB[a,l]P initiation-promotion study, the co-treated group had significantly fewer papillomas per mouse than mice treated with DB[a,l]P alone as initiator. Averaging over the times of exposure gave strong evidence that mice co-treated with SRM and DB[a,l]P had a significantly lower level of PAH-DNA adducts than mice treated with DB[a,l]P alone. Western immunoblots showed that both cytochrome P450 (CYP) 1A1 and 1B1 were induced by the SRM. These results are consistent with the hypothesis that two major factors determining the carcinogenic activity of PAH within a complex mixture are (i) the persistence of certain PAH-DNA adducts as well as total adduct levels, and (ii) the ability of the components present in the mixture to inhibit the activation of carcinogenic PAH by the induced CYP enzymes.

Effects of the (-)-anti-11R,12S-dihydrodiol 13S,14R-epoxide of dibenzo.

The tumor suppressor protein p53 plays an important role in recognition of DNA damage and induction of subsequent cell cycle arrest. One of its target genes encodes the protein p21(WAF1), which is involved in mediation of growth arrest after DNA damage has occurred. Dibenzo[a,l]pyrene (DB[a,l]P) is a polycyclic aromatic hydrocarbon which is an exceptionally potent carcinogen. A reactive secondary metabolite of DB[a,l]P, the fjord region (-)-anti-11R,12S-dihydrodiol 13R,14S-epoxide [(-)-anti-DB[a,l]PDE] was used to investigate DNA damage via adduct formation and cell cycle arrest in human diploid fibroblast cell cultures (HDF). Synchronous HDF were exposed to increasing concentrations (0.014, 0.028 and 0.07 microM) of (-)-anti-DB[a,l]PDE and at 1, 12, 24 and 42 h after treatment cell pellets were analyzed for DNA adduct formation and cell cycle arrest. Exposure of HDF to 0.07 microM (-)-anti-DB[a,l]PDE caused a total DNA binding level of 113 pmol adducts/mg DNA (42 h after treatment). G(1) arrest was induced by this treatment, with 91% of the cells remaining in G(1) phase compared with the solvent-treated control cultures (50%) as analyzed by propidium iodide staining and flow cytometry. Further investigation of the percentage of cells in S phase by 5-bromo-2'-deoxyuridine incorporation confirmed the G(1) arrest in HDF treated with 0.07 microM (-)-anti-DB[a,l]PDE, with only 1.5% of the cells moving into S phase compared with 39% in the control 42 h after treatment. Induction of p53 and p21(WAF1) was demonstrated by western blot analysis.

Influence of beta-naphthoflavone on 7,12-dimethylbenz(a)anthracene metabolism, DNA adduction, and tumorigenicity in rainbow trout.

Metabolism, DNA adduction, and tumor induction by 7, 12-dimethylbenz(a)anthracene (DMBA) were examined in cultured trout liver cells and in vivo in trout. Modulating CYP1A1 activity indicated this enzyme plays a significant role in metabolizing DMBA to water-soluble compounds in isolated trout liver cells. The major DMBA metabolites identified in trout liver cells were 10-, 11-, 8,9-, and 5,6-DMBA dihydrodiols, and DMBA, 2- or 3- or 4-phenol; 7-OH-methyl-12-methyl-benz(a)anthracene and 12-OH-methyl-7-methyl-benz(a)anthracene were minor metabolites. A very small amount of DMBA-3,4-dihydrodiol was detected, and polar metabolites, which did not migrate with any DMBA metabolite standards, were observed. Incubating trout hepatocytes with DMBA-3, 4-dihydrodiol produced three prominent, nonpolar adducts indistinguishable from those in mouse embryo cells. However, DMBA-DNA adducts, formed in trout in vivo or in trout liver cells exposed to DMBA, were predominantly more polar than those formed in mouse embryo fibroblasts, and levels of DMBA-DNA adducts formed in trout liver cells were not significantly altered by modulating CYP1A1 activity. No significant repair of DMBA-DNA adducts was detected in cultured trout liver cells over a 48-h period, supporting previous studies indicating that fish are less efficient than mammals in repairing polyaromatic hydrocarbon DNA adducts. Compared to animals receiving DMBA alone, beta-naphthoflavone pretreatment in vivo did not affect hepatic CYP1A1, DMBA-DNA adducts, nor hepatic tumor response; but did significantly reduce tumor response in two other target organs. These results collectively indicate that DMBA bioactivation to DNA-binding metabolites in trout liver cells and mouse embryo cells predominantly involve different metabolic pathways to form the DNA-binding intermediates.

Cytochrome P4501B1 mediates induction of bone marrow cytotoxicity and preleukemia cells in mice treated with 7,12-dimethylbenz[a]anthracene.

Humans are exposed to polycyclic aromatic hydrocarbons (PAHs) through many environmental pollutants, especially cigarette smoke. These chemicals cause a variety of tumors and immunotoxic effects, as a consequence of bioactivation by P-450 cytochromes to dihydrodiol epoxides. The recently identified cytochrome P4501B1 (CYP1B1) bioactivates PAHs but is also a physiological regulator, as evidenced by linkage of CYP1B1 deficiency to congenital human glaucoma. This investigation demonstrates that CYP1B1 null mice are almost completely protected from the acute bone marrow cytotoxic and preleukemic effects of the prototypic PAH 7,12-dimethylbenz[a]anthracene (DMBA). CYP1B1 null mice did not produce the appreciable amounts of bone marrow DMBA dihydrodiol epoxide DNA adducts present in wild-type mice, despite comparable hepatic inductions of the prominent PAH-metabolizing P-450 cytochrome, CYP1A1. Wild-type mice constitutively expressed low levels of bone marrow CYP1B1. These findings suggest that CYP1B1 is responsible for the formation of DMBA dihydrodiol epoxides in the bone marrow. Furthermore, this study substantiates the importance of DMBA dihydrodiol epoxide generation at the site of cancer initiation and suggests that tissue-specific constitutive CYP1B1 expression may contribute to cancer susceptibility in the human population.

Formation of stable DNA adducts and apurinic sites upon metabolic activation of bay and fjord region polycyclic aromatic hydrocarbons in human cell cultures.

Carcinogenic polycyclic aromatic hydrocarbons (PAH), such as benzo[a]pyrene (B[a]P), 7,12-dimethylbenz[a]anthracene (DMBA), and dibenzo[a,l]pyrene (DB[a,l]P), are metabolically activated to electrophilically reactive bay or fjord region diol epoxides that bind to the exocyclic amino groups of purine bases in DNA to form stable adducts. In addition, it has been reported that these PAH can be enzymatically oxidized to yield radical cations that form apurinic (AP) sites in DNA via depurinating adducts. The formation of stable adducts and AP sites in DNA of human cells exposed to PAH was examined in cytochrome P450 (P450)-expressing mammary carcinoma MCF-7 cells and in leukemia HL-60 cells, which display a high peroxidase but no P450-mediated activity, after exposure to these PAH. Stable DNA adducts were assessed by (33)P-postlabeling/HPLC analysis, and the induction of AP sites in DNA was analyzed by an aldehyde reactive probe (ARP) and a slot blot method. After exposure for 4 h, the levels of stable DNA adducts were comparable in MCF-7 cells treated with B[a]P and DMBA, but significantly lower than those observed in MCF-7 cells treated with the stronger carcinogen DB[a,l]P. While the levels of stable adducts increased more than 10-fold (B[a]P and DMBA) or 100-fold (DB[a,l]P) after exposure for 24 h, the levels of AP sites remained low after both treatment periods. Thus, the levels of stable adducts were approximately 5-fold higher than the levels of AP sites after treatment with B[a]P or DMBA and more than 100-fold higher in cells exposed to DB[a,l]P for 24 h. None of these carcinogenic PAH formed detectable levels of stable DNA adducts or AP sites in HL-60 cells. The results demonstrate that metabolic activation of B[a]P, DMBA, and DB[a,l]P is catalyzed by P450 enzymes leading to diol epoxides that form predominantly stable DNA adducts but only low levels of AP sites.

Multiple sclerosis, dental caries and fillings: a case-control study.

OBJECTIVES: To investigate the association between multiple sclerosis, dental caries, amalgam fillings, body mercury and lead. DESIGN: Matched case-control study. SETTING: Leicestershire in the years 1989-1990. SUBJECTS: Thirty-nine females with multiple sclerosis (of recent onset) were matched with 62 controls for age, sex and general practitioner. METHODS: Home interview of cases and controls within which there was an assessment of the DMFT index and blood and urine mercury and lead levels. RESULTS: The odds of being a MS case increased multiplicatively by 1.09 (95% CI 1.00, 1.18) for every additional unit of DMFT index of dental caries. This represents an odds ratio of 1.213 or a 21% increase in risk of MS in relation to dental caries in this population. There was no difference between cases and controls in the number of amalgam fillings or in body mercury or lead levels. There was a significant correlation between body mercury levels and the number of teeth filled with amalgam (controls: r = +0.430, P = 0.006, cases: r = +0.596, P = 0.001). CONCLUSION: There was evidence of excess dental caries among MS cases compared with the controls. This finding supports the strong geographical correlation between the two diseases. A further study of this association is recommended.

Cancer initiation by polycyclic aromatic hydrocarbons results from formation of stable DNA adducts rather than apurinic sites.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants with high carcinogenic potencies that have been linked to the etiology of human cancers through their presence in cigarette smoke and environmental mixtures. They are metabolically activated in cells by cytochrome P450 enzymes and/or peroxidases to reactive intermediates that damage DNA. One pathway of activation forms dihydrodiol epoxides that covalently bind to exocyclic amino groups of purines in DNA to form stable adducts. Another pathway involves formation of radical cations that bind to the N7 or C8 of purines to form unstable adducts that depurinate to leave apurinic (AP) sites in DNA. In the present study the proportions of stable DNA adducts and AP sites formed by the carcinogenic PAHs dibenzo[a,l]-pyrene (DB[a,l]P), 7,12-dimethylbenz[a]anthracene (DMBA), and benzo[a]pyrene (B[a]P) have been investigated in a target tissue for carcinogenesis, mouse epidermis. After topical application of the PAHs on the skin of female SENCAR mice epidermal DNA was isolated and the formation of stable DNA adducts was measured by (33)P-postlabeling and HPLC analysis. AP sites in DNA were measured with an aldehyde reactive probe in a slot-blot assay. At both 4 and 24 h after exposure, DB[a,l]P formed significantly higher amounts of stable DNA adducts than DMBA, and B[a]P exhibited the lowest level of binding. In contrast, the number of AP sites present in mice treated with these PAHs was in the order: DMBA > B[a]P >> DB[a,l]P. The level of AP sites was significantly lower than the level of stable adducts for each PAH. The most potent carcinogen, DB[a,l]P, induced the highest level of stable adducts and the lowest level of AP sites in epidermal DNA. These results indicate that stable DNA adducts rather than AP sites are responsible for tumor initiation by carcinogenic PAHs.