Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy to diagnose osteoarthritis in equine serum.

BACKGROUND: Reliable and validated biomarkers for osteoarthritis (OA) are currently lacking. OBJECTIVES: To develop an accurate and minimally invasive method to assess OA-affected horses and provide potential spectral markers indicative of disease. STUDY DESIGN: Observational, cross-sectional study. METHODS: Our cohort consisted of 15 horses with OA and 48 without clinical signs of the disease, which were used as controls. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy was used to investigate serum samples (50 µL) collected from these horses. Spectral processing and multivariate analysis revealed differences and similarities, allowing for detection of spectral biomarkers that discriminated between the two cohorts. A supervised classification algorithm, namely principal component analysis coupled with quadratic discriminant analysis (PCA-QDA), was applied to evaluate the diagnostic accuracy. RESULTS: Segregation between the two different cohorts, OA-affected and controls, was achieved with 100% sensitivity and specificity. The six most discriminatory peaks were attributed to proteins and lipids. Four of the spectral peaks were elevated in OA horses, which could be potentially due to an increase in lipids, protein expression levels and collagen, all of which have been previously reported in OA. Two peaks were found decreased and were tentatively assigned to the reduction of proteoglycan content that is observed during OA. MAIN LIMITATIONS: The control group had a wide range of ages and breeds. Presymptomatic OA cases were not included. Therefore, it remains unknown whether this test could also be used as an early diagnostic tool. CONCLUSIONS: This spectrochemical approach could provide an accurate and cost-effective blood test, facilitating point-of-care diagnosis of equine OA.

MGMT promoter hypermethylation and K-RAS, PTEN and TP53 mutations in tamoxifen-exposed and non-exposed endometrial cancer cases.

BACKGROUND: Tamoxifen has anti-oestrogenic and anti-tumour activity in the breast, but is oestrogenic and carcinogenic in the endometrium. It can induce experimental tumours by both hormonal and DNA-damaging mechanisms, but its carcinogenic mode of action in human endometrium remains unclear. METHODS: We investigated whether an epigenetic mechanism, involving promoter hypermethylation of the gene for the DNA repair enzyme MGMT (O6-methylguanine DNA methyltransferase), was associated with K-RAS, TP53 and PTEN mutations in endometrial tumours from women treated with tamoxifen (TAM, n=30) or unexposed to the drug (EC, n=38). RESULTS: There were significant (P<0.05) differences in tumour grade between the TAM and EC groups, with more favourable morphology in the latter. K-RAS mutations, predominantly G>A, occurred in small numbers in both groups. TP53 mutations were of mainly A>G, C>T and indel modifications in both groups, but more frequent in TAM cases. PTEN mutations dominated in EC tumours and were of the type that has large impact on protein function, such as indel or nonsense mutations. These observations alongside the mutational spectrum in PTEN suggest that the malignancies arise from different backgrounds, hence pointing to an effect of tamoxifen. Both groups displayed MGMT promoter hypermethylation. This coincided with mutations more frequently in the TAM (78%) than in the EC (50%) group, even though there were significantly (P<0.05) fewer mutations and methylations in TAM cases. CONCLUSIONS: Although the difference in coincidence did not reach significance with the current sample size, the findings suggest that epigenetic processes may play a role in the way tamoxifen induces endometrial cancer.

Dynamic modeling of α-synuclein aggregation in dopaminergic neuronal system indicates points of neuroprotective intervention: experimental validation with implications for Parkinson's therapy.

Protein aggregation is the major pathological hallmark seen in neurodegenerative disorders such as Parkinson's disease (PD). Alpha-synuclein (αS) is the main component of protein aggregates that form Lewy bodies (LBs) in PD and dementia with LBs. There have been several attempts to intervene in the process of expression, modification, clearance, and aggregation of αS as a therapeutic strategy toward neuroprotection. In this study, we have employed a novel, predictive, system level approach in silico to study four different strategies of anti-aggregation therapies: (a) reduction in αS modifications such as phosphorylation, nitration, or truncation in an approach called "seed clearance;" (b) "anti-oligomerization" approach through blocking the early oligomers formation; (c) "oligomers clearance" process by increasing its lysosomal degradation; and (d) "anti-aggregation" that involves prevention of aggregate formation at a later stage. These strategies were tested in a virtual dopaminergic neuronal system triggered by overexpression (OE) of mutant αS-A53T with or without rotenone (Rot)-induced oxidative stress. The results were compared by analyzing markers related to various end points such as oxidative stress, dopamine (DA) metabolism, proteasome function, survival and apoptosis. The experimental system and anti-oligomerization strategies were recapitulated in vitro in M17 dopaminergic cells overexpressing mutant αS-A53T triggered with Cu(II)-mediated oxidative stress, and the experimental data prospectively corroborated with the predictive results. Through this analysis, we found that intervention in the early part of the aggregation pathway by prevention of oligomer formation and increased clearance is indeed a good neuroprotective strategy, whereas anti-aggregation efforts to break up the aggregate at later stages has negative effects on the system.

Infrared spectroscopy with multivariate analysis to interrogate endometrial tissue: a novel and objective diagnostic approach.

BACKGROUND: Endometrial cancer is the most common gynaecological malignancy in the United Kingdom. Diagnosis currently involves subjective expert interpretation of highly processed tissue, primarily using microscopy. Previous work has shown that infrared (IR) spectroscopy can be used to distinguish between benign and malignant cells in a variety of tissue types. METHODS: Tissue was obtained from 76 patients undergoing hysterectomy, 36 had endometrial cancer. Slivers of endometrial tissue (tumour and tumour-adjacent tissue if present) were dissected and placed in fixative solution. Before analysis, tissues were thinly sliced, washed, mounted on low-E slides and desiccated; 10 IR spectra were obtained per slice by attenuated total reflection Fourier-transform IR (ATR-FTIR) spectroscopy. Derived data was subjected to principal component analysis followed by linear discriminant analysis. Post-spectroscopy analyses, tissue sections were haematoxylin and eosin-stained to provide histological verification. RESULTS: Using this approach, it is possible to distinguish benign from malignant endometrial tissue, and various subtypes of both. Cluster vector plots of benign (verified post-spectroscopy to be free of identifiable pathology) vs malignant tissue indicate the importance of the lipid and secondary protein structure (Amide I and Amide II) regions of the spectrum. CONCLUSION: These findings point towards the possibility of a simple objective test for endometrial cancer using ATR-FTIR spectroscopy. This would facilitate earlier diagnosis and so reduce the morbidity and mortality associated with this disease.

Low-dose nesiritide in human anterior myocardial infarction suppresses aldosterone and preserves ventricular function and structure: a proof of concept study.

BACKGROUND: B-type natriuretic peptide (BNP, nesiritide) has anti-fibrotic, anti-hypertrophic, anti-inflammatory, vasodilating, lusitropic and aldosterone-inhibiting properties but conventional doses of BNP cause hypotension, limiting its use in heart failure. OBJECTIVE: To determine whether infusion of low-dose BNP within 24 h of successful reperfusion for anterior acute myocardial infarction (AMI) would prevent adverse left ventricular (LV) remodelling and suppress aldosterone. METHODS: A translational proof-of-concept study was carried out to determine tolerability and biological activity of intravenous BNP at 0.003 and 0.006 microg/kg/min, without bolus started within 24 h of successful reperfusion for anterior AMI. 24 patients with first anterior wall ST elevation AMI and successful revascularisation were randomly assigned to receive 0.003 (n = 12) or 0.006 (n = 12) microg/kg/min of IV BNP for 72 h in addition to standard care during hospitalisation for anterior AMI. RESULTS: Baseline characteristics, drugs and peak cardiac biomarkers for myocardial damage were similar between both groups. Infusion of BNP at 0.006 microg/kg/min resulted in greater biological activity than infusion at 0.003 microg/kg/min as measured by higher mean (SEM) plasma cGMP levels (8.6 (1) vs 5.5 (1) pmol/ml, p<0.05) and suppression of plasma aldosterone (8.0 (2) to 4.6 (1) ng/dl, p<0.05), which was not seen in the 0.003 microg/kg/min group. LV ejection fraction (LVEF) improved significantly from baseline to 1 month (40 (4)% to 54 (5)%, p<0.05) in the 0.006 group but not in the 0.003 group. Infusion of BNP at 0.006 microg/kg/min was associated with a decrease of LV end-systolic volume index (61 (9) to 43 (8) ml/m(2), p<0.05) at 1 month, which was not seen in the 0.003 group. No drug-related serious adverse events occurred in either group. CONCLUSIONS: 72 h infusion of low BNP at the time of anterior AMI is well tolerated and biologically active. Patients treated with low-dose BNP had improved LVEF and smaller LV end-systolic volume at 1 month.

Use of tissue ink to maintain identification of individual cores on needle biopsies of the prostate.

BACKGROUND: There is an increasing necessity to extract the maximum amount of information, beyond even a cancer diagnosis, from prostate biopsies. Thus, maintaining site-specific information regarding individual biopsy cores might be critical. AIM: To evaluate the applicability of employing tissue ink to maintain the identity of individual prostatic biopsy cores. METHOD: In this ongoing study, 12 core prostate biopsy specimens are sent to the laboratory in individual pots labelled according to anatomical site. The specimens are placed in two separate multi-compartment cassettes. They are inked with different colours to identify the site of origin from each lobe. The cassettes are then processed with a single paraffin block for each side; the six cores from each side can be mounted on a single slide. RESULTS: The different colours used adhere well to the biopsy cores, thus maintaining the identity of each core. Six cores from each side are embedded in a single paraffin block and examined on a single slide, making it cost-effective, while maintaining high quality, accurate histopathological information. CONCLUSION: Differential inking of prostate biopsy cores is an easily applicable method that is cost-effective and provides tumour location information. Prostate biopsy data archived to maintain individual core information might be used to determine applicability of such information to predict extra-capsular extension by correlating with imaging and radical prostatectomy findings, and for treatment planning.

Differential gene expression in the peripheral zone compared to the transition zone of the human prostate gland.

Gene expression profiles may lend insight into whether prostate adenocarcinoma (CaP) predominantly occurs in the peripheral zone (PZ) compared to the transition zone (TZ). From human prostates, tissue sets consisting of PZ and TZ were isolated to investigate whether there is a differential level of gene expression between these two regions of this gland. Gene expression profiling using Affymetrix Human Genome U133 plus 2.0 arrays coupled with quantitative real-time reverse transcriptase-PCR was employed. Genes associated with neurogenesis, signal transduction, embryo implantation and cell adhesion were found to be expressed at a higher level in the PZ. Those overexpressed in the TZ were associated with neurogenesis development, signal transduction, cell motility and development. Whether such differential gene expression profiles may identify molecular mechanisms responsible for susceptibility to CaP remains to be ascertained.

Diversity of TMPRSS2-ERG fusion transcripts in the human prostate.

TMPRSS2-ERG gene fusions have recently been reported to be present in a high proportion of human prostate cancers. In the current study, we show that great diversity exists in the precise structure of TMPRSS2-ERG hybrid transcripts found in human prostates. Fourteen distinct hybrid transcripts are characterized, each containing different combinations of sequences from the TMPRSS2 and ERG genes. The transcripts include two that are predicted to encode a normal full-length ERG protein, six that encode N-terminal truncated ERG proteins and one that encodes a TMPRSS2-ERG fusion protein. Interestingly, distinct patterns of hybrid transcripts were found in samples taken from separate regions of individual cancer-containing prostates, suggesting that TMPRSS2-ERG gene fusions may be arising independently in different regions of a single prostate.

Lycopene inhibits DNA synthesis in primary prostate epithelial cells in vitro and its administration is associated with a reduced prostate-specific antigen velocity in a phase II clinical study.

Interest in lycopene has focused primarily on its use in the chemoprevention of prostate cancer (CaP); there are few clinical trials involving men with established disease. In addition, most data examining its mechanism of action have been obtained from experiments using immortal cell lines. We report the inhibitory effect(s) of lycopene in primary prostate epithelial cell (PEC) cultures, and the results of a pilot phase II clinical study investigating whole-tomato lycopene supplementation on the behavior of established CaP, demonstrating a significant and maintained effect on prostate-specific antigen velocity over 1 year. These data reinforce the justification for a large, randomized, placebo-controlled study.

Primary cultures of prostate cells and their ability to activate carcinogens.

Differences in the incidence of prostate cancer (CaP) amongst different migrant populations point to causative agents of dietary and/or environmental origin. Prostate tissues were obtained following transurethral resection of the prostate (TURP) or radical retropubic prostatectomy. After surgery, TURP-derived or tumour-adjacent tissue fragments were minced in warm PFMR-4A medium (37 degrees C) and suspensions pipetted into collagen-coated petri dishes. Non-adherent material was removed by washing with fresh medium after 12 h. Adhered cells subsequently reacted positively with monoclonal antibodies to prostate specific antigen (PSA). PSA was also detected in the medium. The genotoxicities of the chemical carcinogens 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP), its N-hydroxy metabolite (N-OH-PhIP) and benzo[a]pyrene (B[a]P) in adherent cell populations from different donors (n=8) were examined. Cells were treated in suspension for 30 min at 37 degrees C in the presence of the DNA repair inhibitors hydroxyurea (HU) and cytosine arabinoside (ara-C). DNA single-strand breaks were detected in cells by the alkaline single cell-gel electrophoresis ('Comet') assay and quantified by measuring comet tail length (CTL) in microm. All three carcinogens induced dose-related increases in CTLs (P<0.0001) in cells from four donors 24 h post-seeding. However, in cells from a further two donors the genotoxic effects of PhIP, N-OH-PhIP and B[a]P were much less apparent after 48 h than after 24 h in culture. After 96 h in culture, cells from these donors appeared to be resistant to the comet-forming activity of the compounds. However, B[a]P-DNA adducts were still measurable by (32)P-postlabelling for up to 14 days following a 24-h exposure to 50 microM B[a]P in adhered cells from another two donors. This study shows that primary cultures of cells derived from the prostate can activate members of two classes of chemical carcinogens. Further development may provide a robust model system in which to investigate the aetiology of CaP.

Morphological transformation of C3H/M2 mouse fibroblasts by, and genotoxicity of, extracts of human milk.

Breast cancer may be initiated by environmental/dietary agents and human milk may act as an ex vivo indicator of in vivo exposure of mammary epithelial cells to genotoxins. Extracts of human milk from UK-resident women (n=7) were tested for their abilities to morphologically transform C3H/M2 mouse fibroblasts. Genotoxicities were assessed in the Salmonella typhimurium reverse-mutation assay in the presence of S9 using strains TA1538 and YG1019, and in metabolically-competent human MCL-5 cells with the micronucleus and with the alkaline single cell gel electrophoresis (comet) assays. Two of the seven extracts were inactive in the transformation assay both in the presence or absence of S9, two appeared to be equally transforming either in the presence or absence of S9, and two other extracts induced increased transformation frequencies in the presence of S9. A seventh extract, tested only in the absence of S9, was inactive. Extracts were either active or inactive in at least three of the four tests applied. Four extracts were active or inactive in all four tests. The results suggest that human milk could be used as a resource for investigations of the as-yet-unidentified transforming agents previously detected in mammary lipid.

Formation of Strecker aldehydes and pyrazines in a fried potato model system.

Sugars and amino acids were removed from potato slices by soaking in water and ethanol. They were then infused with various combinations of sugars (glucose and/or fructose) and amino acids (asparagine, glutamine, leucine, isoleucine, phenylalanine, and/or methionine) and fried. Volatile compounds were trapped onto Tenax prior to gas chromatography-mass spectrometry. Relative amounts of compounds (relative to the internal standard) and relative yields (per mole of amino acid infused into the slices) were determined. Amounts of 10 pyrazines, 4 Strecker aldehydes, and 4 other compounds were monitored. Relative amounts and relative yields of compounds varied according to the composition of the system. For the single amino acid-glucose systems, leucine gave the highest relative amount and relative yield of its Strecker aldehyde. Asparagine and phenylalanine gave the highest total relative amount and total relative yield, respectively, of pyrazines. In the system containing all of the amino acids and glucose, the relative amount of 3-methylbutanal was higher, whereas the amounts of the other monitored Strecker aldehydes were lower. Most of the relative amounts of individual pyrazines were lower compared to the glucose-asparagine system, whereas the total relative yield of pyrazines was lower, compared to all of the single amino acid-glucose mixtures. Addition of fructose to the mixed amino acid-glucose model system generated Strecker aldehydes and pyrazines in ratios that were more similar to those of untreated potato chips than to those from the same system but without fructose. Both the sugars and the amino acids present in potato are crucial to the development of flavor compounds in fried potato slices.

Genotoxicity of human breast milk from different countries.

Dietary and/or environmental factors appear to play a key role in the international variations that exist in breast cancer incidence. The genotoxicity of breast milk extracts is being examined as a possible indicator of in vivo exposure of mammary epithelial cells to DNA-damaging agents. Breast milk samples were obtained from the UK (n = 32), a high risk country, and from Hong Kong (n = 10), India (n = 20) and Singapore (n = 20), countries of lower breast cancer incidence. The abilities of breast milk extracts to induce DNA damage detected as single-strand breaks (SSBs) in the alkaline Comet assay and to induce micronuclei in MCL-5 cells and mutations in Salmonella typhimurium YG1019 were investigated. In the Comet assay 18 of 32 (56%) UK samples induced significant increases in DNA SSBs compared with 2 of 10 (20%), 5 of 20 (25%) and 8 of 20 (40%) of the samples from Hong Kong, India and Singapore, respectively. The proportion of positive samples was significantly higher in the UK group than in the combined low breast cancer incidence group and significantly higher than in the Indian group (P < 0.05, Fisher's exact test). In the micronucleus assay 9 of 32 (28%) UK samples showed significant activity compared with 0 of 10 (0%), 2 of 20 (10%) and 3 of 20 (15%) of the samples from Hong Kong, India and Singapore, respectively. Extracts of all the aforementioned milk samples were also tested for bacterial mutagenicity. Nine of 32 (28%) UK samples induced significant activity with a dose-response effect. Although activity was detected in samples from the other countries, comparable dose-response data could not be obtained because of a lack of material. This pilot study suggests that genotoxic components occur more frequently in UK breast milk than in milk from some other countries with a lower incidence of cancer. More work is required to confirm these initial findings and to examine their relevance to variations in breast cancer incidence.

Genotoxins and the initiation of sporadic breast cancer.

Breast cancer is the most frequently diagnosed female malignancy world-wide. The aetiology of the majority of cases remains obscure and the only genotoxin as yet known to initiate breast cancer is ionizing radiation. High penetrance susceptibility genes probably account for no more than 5-10% of cases. The breast, which consists of 70-90% adipose tissue, has a unique morphological structure. Dispersed within it are the functional elements that are lined with cancer-susceptible epithelial cells. Numerous dietary and/or environmental fat-soluble compounds are known to be rodent mammary carcinogens. Extracts of lipid obtained following collagenase digestion of elective reduction mammoplasty tissues from UK resident women showed activity in short-term genotoxicity assays in 40% of cases. More active lipid extracts tended to come from donors whose human mammary epithelial cells (HMECs) exhibited pre-existing DNA single-strand breaks (SSBs). Lipid extracts also induced morphological transformation of mammalian cells in vitro. To increase cohort size, extracts of UK breast milk were examined for genotoxicity and similar activity profiles were observed. Viable cells, a large percentage of which were HMECs, were recovered from breast milk and examined for pre-existing SSBs and for the ability of the donor's own milk extract to induce SSBs. Again, donors whose untreated cells contained the most SSBs tended to yield genotoxic breast milk extracts. Breast milk cells were also able to activate rodent mammary carcinogens to DNA-damaging species. These studies provide good evidence for in vivo exposure of HMECs to genotoxic agents years before the peak in occurrence of sporadic breast cancer. Work is in progress to characterize these agents and to determine their possible role in breast cancer aetiology.

Activation of genotoxins to DNA-damaging species in exfoliated breast milk cells.

Exfoliated cells, isolated from breast milk samples donated by UK-resident women (n=15), were incubated, either immediately or after culture for 7 days, with one of a series of genotoxins, either in the presence or absence of the DNA-repair inhibitors, hydroxyurea (HU), and cytosine arabinoside (ara-C). The numbers of DNA single-strand breaks induced were then assessed as comet tail length (CTL) (microm) using the alkaline single cell-gel electrophoresis ('Comet') assay; cell viability was measured by trypan blue exclusion. The heterocyclic aromatic amines (HAAs) (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) (0.4 mM), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) (1.67 mM), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) (1.77 mM)), a polycyclic aromatic hydrocarbon (benzo[a]pyrene (B[a]P) (0.36 mM)), a nitro-polycyclic aromatic hydrocarbon (1-nitropyrene (1-NP) (1.84 mM)) and aromatic amines (o-toluidine (0.85 mM), p-chloroaniline (0. 71 mM)) each induced statistically significant (P<0.0001, Mann-Whitney test) increases in median CTLs in breast milk cells from all the donors examined when incubated (30 min, 37 degrees C) in the presence of HU/ara-C. In some cases, these compounds were also active in the absence of the repair inhibitors. There were marked variations in comet formation between donors and between genotoxins. Cell culture appeared to increase the epithelial cell proportion and cultured cells retained their ability to activate genotoxins. The results suggest that breast milk is a valuable source of human mammary cells for the study of the metabolic activation of possible carcinogens.

Metabolic activation of carcinogens and expression of various cytochromes P450 in human prostate tissue.

Epidemiological evidence suggests a link between meat consumption and prostate cancer. In this study, benign prostatic hyperplasia tissues, obtained by transurethral resection or radical retropubic prostatectomy from UK-resident individuals (n = 18), were examined for CYP1 expression and for their ability, in short-term organ culture, to metabolically activate carcinogens found in cooked meat. Semi-quantitative RT-PCR analysis of CYP1 expression detected CYP1A2 mRNA transcripts in the prostates of four individuals, as well as mRNA transcripts from CYP1A1 and CYP1B1. The compounds tested for metabolic activation were 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (PhIP; 500 microM, n = 9) and its metabolite N:-hydroxy PhIP (20 microM, n = 8), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ; 500 microM, n = 6) and benzo[a]pyrene (B[a]P; 50 microM, n = 5). After incubation (PFMR medium, 22 h, 37 degrees C), DNA was isolated from tissue fragments and DNA adducts were detected and quantified by (32)P-postlabelling analysis. DNA adduct formation was detected in all samples incubated with PhIP (mean, adducts per 10(8) nucleotides), N:-hydroxy-PhIP (2736/10(8)) or B[a]P (1/10(8)). IQ-DNA adducts were detected in 5/6 tissues (mean, 1/10(8)). The CYP1 inhibitor alpha-naphthoflavone (10 microM) reduced B[a]P-DNA adduct formation in tissues from two individuals by 96 and 64%, respectively. This pilot study shows that human prostate tissue can metabolically activate 'cooked meat' carcinogens, a process that could contribute to prostate cancer development.

Cell transformation and genotoxicity induced by bis(2, 3-dichloro-1-propyl) ether.

Bis(dichloropropyl) ether isomers have been identified in a petrochemical plant effluent through a toxicity identification evaluation study in the United States. They have also been observed in the microgram per liter range along one of the largest rivers in Europe, the Elbe River. In the present investigation, the genotoxic and transforming activity of a bis(dichloropropyl) ether isomer, bis(2,3-dichloro-1-propyl) ether, was assayed in vitro. The results demonstrate that bis(2,3-dichloro-1-propyl) ether is a potent mutagen in Salmonella typhimurium strains TA 100, TA 1535, and to a lesser extent in strain TA 98, but only when tested in the presence of a metabolic activation system (S9 mix). We have also investigated the induction of micronuclei by bis(2,3-dichloro-1-propyl) ether in the metabolically competent cell line, MCL-5. A linear, dose-dependent increase in micronuclei was observed following exposure to bis(2,3-dichloro-1-propyl) ether. The DNA strand-breaking capacity of this chemical was assessed in the alkaline single-cell gel electrophoresis ("comet") assay with MCL-5 cells. Bis(2,3-dichloro-1-propyl) ether clearly induced DNA strand breaks in the 4.5-45.5 microg/ml dose range. The ether also induced malignant transformation in C3H/M2 mouse fibroblasts after metabolic activation (S9 mix). Thus, it must be suspected that bis(2, 3-dichloro-1-propyl) ether may possess a carcinogenic potential. Since the compound along with its isomers is present in considerable concentrations in surface water, their elimination is a matter of significant public concern.

DNA damage in human breast milk cells and its induction by 'early' and 'late' milk extracts.

Environmental and dietary factors are thought to be significant in breast cancer aetiology. The alkaline single-cell gel electrophoresis ('Comet') assay was used to examine breast milk cells for DNA damage and to measure the activity of extracts of the milk in causing such damage. UK-resident women were recruited as donors (n = 16) and provided 'early' ( approximately 4 weeks post-partum) and/or 'late' ( approximately 4 months post-partum) milk samples. Cells (79-94% viable, trypan blue exclusion) were either examined immediately for DNA damage or were cultured for 1 week prior to treatment with a breast milk extract. DNA damage in the form of single-strand breaks was quantified as comet tail length (CTL). Cell preparations examined immediately exhibited interindividual variation in median CTL (range 2.0-40.0 microm) with or without the DNA repair inhibitors hydroxyurea (HU) and cytosine arabinoside (ara-C). DNA damage decreased following culture, suggesting either DNA repair or death of DNA-damaged cells. Some donors' breast milk extracts induced DNA damage in their cultured cells and increases in median CTL were significantly greater with HU/ara-C (range 4.0-72.5 microm) than without (range 2.5-27.5 microm). Genotoxicity occurred without cytotoxicity (81-97% viability after treatment). Comparisons between cells and extracts from 'early' and 'late' milk samples did not support the idea of a progressive clearance of genotoxins from mammary lipid during lactation. Donors whose untreated cells contained the most DNA damage tended to yield genotoxic breast milk extracts. Cells isolated from milk activated the rodent mammary carcinogens o-toluidine and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). The relevance of genotoxic exposures to breast cancer initiation requires further investigation.

The influence of dietary and environmental factors on prostate cancer risk.

Prostate cancer is the most common malignancy found in males and one of the most common causes of cancer death. The epidemiology implicates environmental and nutritional factors in the initiation and progression of the disease. Identification of these factors would allow chemoprevention strategies to be tested. Potent mutagenic and carcinogenic heterocyclic amines and polycyclic aromatic hydrocarbons are produced in cooked meat, and following metabolic activation some of them are strongly associated with prostate carcinogenesis in rodents. Primary cell cultures of human prostate epithelial cells were obtained from patients undergoing transurethral resection of the prostate. Metabolic activation of the cooked food carcinogens 2-amino-1-methyl-6-phenylimidazo- [4,5-b]pyridine (PhIP) and benzo[a]pyrene (B[a]P) was examined and resultant DNA damage (single strand breaks) measured using the Comet assay. Increased concentrations of carcinogen were associated with increased DNA damage and comet tail length compared to controls. Prostate Cancer and Prostatic Diseases (2000) 3, 256-258

The DNA repair inhibitors hydroxyurea and cytosine arabinoside enhance the sensitivity of the alkaline single-cell gel electrophoresis ('comet') assay in metabolically-competent MCL-5 cells.

We have found previously that the metabolically-competent human MCL-5 cell line did not appear to be usefully sensitive to the DNA-damaging effects of several carcinogens, as measured by the alkaline single-cell gel electrophoresis ('comet') assay. We therefore sought to increase its sensitivity by inhibiting DNA repair during exposure to test compounds, using 10 mM hydroxyurea (HU) and 1.8 mM cytosine arabinoside (ara-C), which inhibit DNA resynthesis during nucleotide excision repair. The following compounds were tested, using a 30-min exposure, in the absence or presence of HU/ara-C: 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4, 8-DiMeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-9H-pyrido[2,3-b]indole (A[alpha]C), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA[alpha]C), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), benzo[a]pyrene (B[a]P), 3-methylcholanthrene (3-MCA), 7, 12-dimethylbenz[a]anthracene (DMBA), 1-nitropyrene (1-NP), 2-nitrofluorene (2-NF), aniline, o-toluidine, benzene, lindane, bleomycin, cisplatin, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), sodium chromate, chromic chloride, and diethylstilboestrol (DES). We made the following observations. The background level of comet formation was reasonably constant over several months and was increased only slightly, but significantly, in the presence of the DNA-repair inhibitors. All compounds that induced comet formation did so without appreciable cytotoxicity as assessed by trypan blue exclusion. Of the compounds tested, the heterocyclic amines and polycyclic aromatic hydrocarbons (with the exceptions of PhIP and B[a]P) failed to induce convincing levels of comet formation in the absence of repair inhibitors. In their presence the heterocyclic amines tested induced comet formation (with the exception of 8-MeIQx), with widely differing potencies. 1-NP failed to elicit marked comet formation even in the presence of HU/ara-C. Aniline and o-toluidine produced significant levels of comet formation in the absence of HU/ara-C, but in their presence comet formation was markedly increased. Benzene, lindane, bleomycin, cisplatin, MNNG, sodium chromate and chromic chloride induced comet formation in the absence of HU/ara-C, but, with the exception of cisplatin, their presence enhanced comet formation. Neither sucrose nor DES elicited comet formation under the conditions used in this study. Many more agents need to be tested in order to determine how well the comet assay using MCL-5 cells (or modified versions of it) can distinguish genotoxins from non-genotoxins.