Risk assessment of coumarin using the bench mark dose (BMD) approach: children in Norway which regularly eat oatmeal porridge with cinnamon may exceed the TDI for coumarin with several folds.

Coumarin is a naturally occurring flavouring substance in cinnamon and many other plants. It is known that coumarin can cause liver toxicity in several species, and it is considered a non-genotoxic carcinogen in rodents. By using the bench mark dose approach we re-assessed coumarin toxicity and established a new TDI for coumarin of 0.07 mg/kg bw/day. Oral intake of coumarin is related to consumption of cinnamon-containing foods and food supplements. Cinnamon is a widely used spice in Norway, and can be used as topping on oatmeal porridge. Based on analyses of coumarin in Norwegian foods, intake calculations for children and adults were conducted, and a risk assessment of coumarin in the Norwegian population was performed. Intake estimates of coumarin show that small children eating oatmeal porridge several times a week sprinkled with cinnamon could have a coumarin intake of 1.63 mg/kg bw/day and may exceeding the TDI with several folds. Adults drinking cinnamon-based tea and consuming cinnamon supplements also can exceed TDI. The coumarin intake could exceed the TDI by 7- to 20-fold in some intake scenarios. Such large daily exceedances of TDI, even for a limited time period of 1-2 weeks, cause concern of adverse health effects.

Risk assessment of dietary exposures to compounds that are genotoxic and carcinogenic--an overview.

The process of risk assessment of dietary exposures to genotoxic carcinogens is summarised. Exposures to six genotoxic carcinogens in food (acrylamide, aflatoxin B1, benzo(a)pyrene, dimethylnitrosamine, ethyl carbamate, PhIP) have been used to illustrate the process. The margin of exposure (MOE) approach is seen as a useful method to be used in the risk characterisation step of assessing exposures to genotoxic carcinogens. This approach combines information on animal potency and human exposure, and can be used to indicate levels of concern and also the ranking between various exposures to such agents. Both the T25 and the BMDL10 methods may be used as a reference point. Should a specific MOE value be developed as a cut-off between levels of concern and levels of low concern, the value using T25 data is proposed to be 2.5-times higher than using BMDL10 data. Linear low-dose extrapolation using either T25 or BMDL10, may also be applied. However, it should be understood that this approach should not be interpreted as giving a precise estimate of human risk. For exposures to mutagens in food lacking carcinogenicity data, it is proposed to apply the MOE approach to the lowest effective dose (LED) for in vivo genotoxicity.

Risk assessment of acrylamide in foods.

Daily mean intakes of acrylamide present in foods and coffee in a limited Norwegian exposure assessment study have been estimated to be 0.49 and 0.46 microg per kg body weight in males and females, respectively. Testicular mesotheliomas and mammary gland adenomas have consistently been found in 2-year drinking water rat cancer studies with acrylamide. Acrylamide also shows initiating activity in mouse skin after systemic administration. Since acrylamide is converted to the mutagenic metabolite glycidamide and forms adducts to hemoglobin in rodents and humans, the tumorigenic endpoints in rats were assumed to be an expression of acrylamide genotoxicity. Using the default linear extrapolation methods LED10 and T25, the lifetime cancer hazard after lifelong exposure to 1 microg acrylamide per kg body weight per day, scaled to humans, was calculated to be, on average, 1.3 x 10-3. Using this hazard level and correlating it with the exposure estimates, a lifetime cancer risk related to daily intake of acrylamide in foods for 70 years in males was calculated to be 0.6 x 10-3, implying that 6 out of 10,000 individuals may develop cancer due to acrylamide. For females, the risk values were slightly lower. It must be emphasized that this risk assessment is conservative. A number of processes may result in nonlinearity of the dose-response relationships for acrylamide carcinogenicity in the low-dose region, including detoxication reactions, cell cycle arrest, DNA repair, apoptosis, and immune surveillance. Thus, the true risk levels related to acrylamide intake may be considerably lower.

A simple method for quantitative risk assessment of non-threshold carcinogens based on the dose descriptor T25.

This report provides guidance for using the dose-descriptor T25 from animal studies as a basis for quantitative risk characterisation of non-threshold carcinogens. T25 is presently used within the European Union for setting specific concentration limits for carcinogens in relation to labelling of preparations (formulations). The T25 is defined as the chronic dose rate which will give 25% of the animals tumours at a specific tissue site, after correction for spontaneous incidence, within the standard life-time of that species. The T25 is converted to the corresponding human dose descriptor, HT25, by dividing it with the appropriate scaling factor for interspecies dose scaling based on comparative metabolic rates. Subsequently, the human dose (expressed in mg per kg body-weight per day) is calculated from the available exposure data. The corresponding human life-time cancer risk is then obtained by using linear extrapolation by dividing the exposure dose with the coefficient (HT25/0.25). The results with this new method, which can easily be calculated without computer programmes, are in excellent agreement with results from computer-based extrapolation methods such as the linearised multistage model and the benchmark method using LED10, even though the present method only takes into consideration one single dose-response point. To overcome possible shortcomings of the present method, the estimated life-time risks are proposed to be accompanied by a commentary statement giving an overall evaluation of data that may have bearing on the carcinogenic risk and that may indicate whether the real human risk is likely to be higher or lower than the calculated life-time risk. By using the present guidance and a harmonized set of criteria and default values, the calculation of life-time cancer risk should be transparent and easy to comprehend.

Phosphorylation of connexin43 and inhibition of gap junctional communication in 12-O-tetradecanoylphorbol-13-acetate-exposed R6 fibroblasts: minor role of protein kinase C beta I and mu.

12-O:-tetradecanoylphorbol-13-acetate (TPA) inhibits gap junctional communication in many cell culture systems, but TPA-induced phosphorylation of the gap junction protein connexin43 (Cx43) varies much between systems. We have here studied whether these responses and their sensitivities can be correlated with total protein kinase C (PKC) enzyme activity and if specific PKC isoenzymes are involved. Rat R6 fibroblasts transfected with the cDNA sequence encoding PKC beta I (R6-PKC3) had a total PKC activity 7- to 16-fold higher than the corresponding control cells (R6-C1), depending on the selection pressure (G418 concentration). Still, R6-PKC3 cells were no more sensitive than R6-C1 cells to TPA-induced down-regulation of communication, except at the highest selection pressure (500 micrograms/ml G418). Thus, total PKC activity does not indicate absolute sensitivity of a cell system to TPA-induced suppression of communication, but within a certain cell system increasing PKC activity may enhance the sensitivity to TPA in this respect. The results also suggest that PKC beta I is of minor importance for TPA-induced regulation of communication. Experiments with the Lilly compound 379196, a PKC beta-specific inhibitor, further supported this conclusion. Except for PKC beta I in R6-PKC3 cells, both cell lines contained the TPA-responsive PKC isoenzymes alpha, delta, epsilon and mu. Long-term treatment with TPA caused strong down-regulation of PKC alpha, delta and epsilon, but little down-regulation of PKC mu. Concurrently, the cells became refractory to repeated exposure to TPA, indicating that PKC mu is of minor importance. Experiments with the general PKC inhibitor GF109203X and the PKC alpha (and beta/gamma) inhibitor Gö6976 suggested that both classical (alpha) and novel PKCs (delta and epsilon) might be involved in TPA-induced suppression of intercellular communication, while phosphorylation of Cx43 may mainly be mediated by PKC alpha in the present systems.

Quantitative determination of gap junction intercellular communication using flow cytometric measurement of fluorescent dye transfer.

Gap junction intercellular communication (GJIC) is involved in several aspects of normal cell behaviour, and disturbances in this type of communication have been associated with many pathological conditions. Reliable and accurate methods for the determination of GJIC are therefore important in studies of cell biology. (Tomasetto, C., Neveu, M.J., Daley, J., Horan, P.K. and Sager, R. (1993) Journal of Cell Biology, 122, 157-167) reported some years ago the use of flow cytometer to determine transfer between cells of a mobile dye, calcein, as a measure of cell communication through gap junctions. In spite of this being a method with potential for quantitative and reliable determination of GJIC, it has been modestly used, possibly due to technical difficulties. In the present work we have illustrated several ways to use flow cytometric data to express cell communication through gap junctions. The recipient cells were pre-stained with the permanent lipophilic dye PKH26, and the donor cell population were loaded with the gap junction permeable dye, calcein. We show that the method may be used to measure the effect of chemicals on GJIC, and that the information is reliable, objective and reproducible due to the large number of cells studied. The data may give additional information to that obtained with other methods, since the effect observed will be on the establishment of cell communication as compared to what is observed for microinjection or scrape loading, where the effect is on already established communication. This is probably the reason for the more potent effects of DMSO on GJIC measured by the present method than on already existing GJIC measured by microinjection or quantitative scrape loading. We also show that the problem related to the mobile dye calcein not being fixable with aldehydes will not affect the results as long as the cells are kept on ice in the dark and analysed by flow cytometer within the first hours after formalin cell fixation.

Morphological transformation and effect on gap junction intercellular communication in Syrian hamster embryo cells as screening tests for carcinogens devoid of mutagenic activity.

A large fraction of chemicals observed to cause cancer in experimental animals is devoid of mutagenic activity. It is therefore of importance to develop methods that can be used to detect and study environmental carcinogenic agents that do not interact directly with DNA. Previous studies have indicated that induction of in vitro cell transformation and inhibition of gap junction intercellular communication are endpoints that could be useful for the detection of non-genotoxic carcinogens. In the present work, 13 compounds [chlordane, Arochlor 1260, di(2-ethylhexyl)phthalate, 1,1,1-trichloro-2, 2-bis(4-chlorophenyl)ethane, limonene, sodium fluoride, ethionine, o-anisidine, benzoyl peroxide, o-vanadate, phenobarbital, 12-O-tetradecanoylphorbol 13-acetate and clofibrate] have been tested for their ability to induce morphological transformation and affect intercellular communication in Syrian hamster embryo cells. The substances were selected on the basis of being proven or suspected non-genotoxic carcinogens, and thus difficult to detect in short-term tests. The data show that nine of the 13 compounds induced morphological transformation, and seven of the 13 inhibited intercellular communication in hamster embryo cells. Taken together, 12 of the 13 substances either induced transformation or caused inhibition of communication. The data suggest that the combined use of morphological transformation and gap junction intercellular communication in Syrian hamster embryo cells may be beneficial when screening for non-genotoxic carcinogens.

[How to enforce the prohibition of sales of tobacco to minors?]. / Hvordan håndheves 18-årsgrensen for kjøp av tobakk?

The introduction in 1996 of a ban on the sale of tobacco to persons under the age of 18 in Norway does not seem to have reduced the extent of smoking among minors. One reason may be that Norway's 20,000 tobacconists have not respected the age limit. A representative sample of households was randomly selected from a database containing all telephone numbers in Norway. Of the 6,135 households contacted, 2,054 households contained young people aged 13 to 20 years. Of these, 1,011 persons participated in the telephone survey. 75% of the tobacco smoked by 13-17-year-olds is bought by the minors themselves or by other minors. 70% of smokers under the age of 18 report not being asked how old they were when they bought or tried to buy tobacco. Only 48% had been denied purchase of tobacco during the last three months. The systematic anti-smoking efforts being instituted in the schools would be much more effective if they were backed up by the tobacconists through effective enforcement of the 18-year age limit for the purchase of tobacco.

[How to reduce illegal sales of tobacco to minors?]. / Hvordan redusere salg av tobakk til mindreårige?

Of the tobacco consumed by young people between the ages of 13 and 17 in Norway, 75% is bought by minors. The Ministry of Health has requested the tobacconist's trade association to improve the enforcement of the 18-year age limit for the purchase of tobacco. After identifying 122 scientific articles through searches in Medline and Sociological Abstracts, we have reviewed the scientific literature on the effects of compliance-enhancing measures designed by the authorities in other countries. Four types of measures, including sanctions against tobacconist, have been used to improve age limit compliance. Voluntary agreements lead to higher tobacconist compliance; however, 20% of them still sell tobacco to minors. This is enough for young people not to report changes in availability or changes in smoking habits. Frequent spot tests, threats of fines or the revoking of licence have led to fewer young smokers. We conclude that the present Norwegian efforts at increasing tobacconist compliance are unlikely to lead to fewer smokers among minors.

Passive smoking, sudden infant death syndrome (SIDS) and childhood infections.

1. A number of cohort and case-control studies have shown clear, dose-related associations between maternal smoking and infant death. The strongest relationships were found when the mother smoked during pregnancy as well as postnatally. Maternal smoking during pregnancy increases the risk for SIDS in most studies, whereas it appears that maternal smoking only postnatally also leads to an increase in risk. In addition, smoking only by the father appears to increase the risk for SIDS, but this is not seen in all studies. 2. Exposure of children to environmental tobacco smoke (ETS) increases the risk of having night cough and respiratory infections (bronchitis, bronchiolitis, pneumonia), especially during the first 2 years of life. An increased risk is also seen in studies not distinguishing between upper and lower respiratory diagnoses. Long-term breastfeeding may have a protective effect on ETS-increased risk of lower respiratory tract illness. One study of older children reports that ETS combined with allergy increased the risk of acute respiratory tract infections above that due to ETS alone. 3. The number of new episodes and duration of otitis media with effusion in young children is positively correlated with ETS exposure. Especially infants with lower birth weights had a high risk of recurrent otitis media during the first year of life when the mother was a heavy smoker. 4. Passive smoking has been reported as a risk factor in meningococcal disease and tuberculosis in young children.

Role of Cx43 phosphorylation and MAP kinase activation in EGF induced enhancement of cell communication in human kidney epithelial cells.

Epidermal growth factor (EGF) has been found to induce enhanced gap junctional intercellular communication (GJIC) in the human kidney epithelial cell line K7. This is in contrast to what is reported for other cell types, which all show decreased GJIC in response to EGF. In the present study it is shown that 12-O-tetradecanoylphorbol-13-acetate (TPA) and EGF induce similar phosphorylation pattern of the gap junction protein connexin43 (Cx43) in K7 cells, although their effects on GJIC are opposite. Tyrosine phosphorylation of a 42 kD protein was observed to be induced concomitantly with phosphorylation of Cx43. EGF was however found to induce only serine phosphorylation of Cx43, indicating that the tyrosine kinase activity of the EGF receptor was not directly affecting the gap junction protein. The 42 kD protein phosphorylated on tyrosine was identified to be a mitogen activated protein (MAP) kinase. Both EGF and TPA was found to activate MAP kinase in these cells. Phosphorylation of Cx43 and enhancement of GJIC in response to EGF occurred with difference in time course. Phosphorylation of Cx43 was completed within 15 min, while the enhanced GJIC appeared 2-3 h later. It is therefore possible that regulation of synthesis or transport of Cx43 is responsible for the increase in GJIC, rather than direct involvement of Cx43 phosphorylation. This is in support of our previous finding that protein synthesis is necessary for EGF induced upregulation of GJIC in K7 cells.

Potency grading in carcinogen classification.

In 1992 the United Nations Conference on Environment and Development decided to harmonize carcinogen classification systems. A proposal for a harmonized classification system is currently being considered by the Organization for Economic Cooperation and Development (OECD). In many countries, classification of a chemical as carcinogenic triggers labeling requirements. Implicit in the labeling requirements are often restrictions on the sale of consumer products and workplace regulations. Many of the current classification systems for carcinogens use a single concentration limit for the minimum concentration of a carcinogen in a preparation (mixture) that requires labeling. For high-potency carcinogens, one concentration limit may not adequately express the hazard, whereas for low-potency carcinogens, one limit may overestimate the hazard caused by the carcinogen in the preparation (mixture). The potency grading system discussed consists of three potency groups: high-, medium-, and low-potency carcinogens. It is envisioned that the different classes will trigger different labeling requirements. In the process of potency grading, a preliminary conclusion as to whether a substance shows high, medium, or low potency is initially based on a tumorigenic dose descriptor. The preliminary potency evaluation may then be modified after due consideration of a number of additional elements. These may include evaluation of the dose-response curve; site-, species-, strain-, and sex-specific activity; mechanisms including genotoxicity; mechanistic relevance to humans; toxicokinetics; and other factors. The potency grading system discussed is applicable to most carcinogen classification systems, including that currently being considered by the OECD.

Effects of peroxisome proliferators and 12-O-tetradecanoyl phorbol-13-acetate on intercellular communication and connexin43 in two hamster fibroblast systems.

Effects of 12-O-tetradecanoyl phorbol 13-acetate (TPA) and the hepatic peroxisome proliferators (HPPs) clofibrate, methyl clofenapate (MCP), di(2-ethylhexyl)phthalate (DEHP) and mono(2-ethylhexyl)phthalate (MEHP) were studied in 2 gap junctional intercellular communication (GJIC) systems, metabolic cooperation in V79 cells and microinjection/dye transfer in Syrian hamster embryo (SHE) cells and V79 cells. TPA inhibited GJIC in both systems but was considerably more potent in V79 cells. SHE cells showed a rapid and transient inhibition of GJIC after exposure to HPPs, with maximal inhibition occurring at 5-15 min. The transient inhibition could be caused by metabolization of the compounds. Clofibrate and MEHP produced strong inhibition of metabolic cooperation in V79 cells at high concentrations, while the effect of MCP and DEHP was lower. However, DEHP, MEHP and clofibrate strongly inhibited dye transfer in V79 cells after a 30 min exposure. Clofibrate also showed a dose- and time-dependent effect on dye transfer in V79 cells. The phosphorylation status of the gap junction protein connexin43 (Cx43) changed minimally in SHE cells after exposure to TPA or HPPs. Cx43 from V79 cells was strongly affected by TPA, but not by HPPs. Immunofluorescence of Cx43 disappeared in both cell types when they were exposed to TPA and MEHP, but not to the other HPPs. Thus, there is no direct correlation between the inhibition of GJIC and changes in the phosphorylation status of Cx43 or the appearance of Cx43 in immunofluorescence experiments. The discrepancies may partly be explained by binding of accessory proteins to Cx43. We point out sequences that may be involved in such binding.

T25: a simplified carcinogenic potency index: description of the system and study of correlations between carcinogenic potency and species/site specificity and mutagenicity.

A simplified carcinogenic potency index, the T25, is proposed as a practical method for the inclusion of potency considerations in carcinogen classification systems. The T25 is the chronic daily dose in mg per kg bodyweight which will give 25% of the animals tumours at a specific tissue site, after correction for spontaneous incidence, within the standard life span of that species. Calculated T25 values of a set of 113 US National Cancer Institute/National Toxicology Program (NC/NTP) carcinogens showed excellent correlation (correlation coefficient 0.96, P < 0.0001) with the carcinogenic potency index TD50 of Peto et al. (1984). The mean of T25 values for 51 transspecies, multiple common site NCI/NTP carcinogens were 10-fold lower than those for 62 NCI/NTP single species, single site carcinogens. For these 113 carcinogens, the mean T25 values were approximately 3-fold lower for agents that were also mutagenic in Salmonella compared to the non-mutagenic agents.

Induction of phosphotyrosine in the gap junction protein, connexin43.

The protein-tyrosine phosphatase inhibitors pervanadate, permolybdate, H2O2, and to a much lesser extent vanadate, increased the amount of cellular phosphotyrosine and induced tyrosine phosphorylation of connexin43 (Cx43) in early passage hamster embryo fibroblasts. The presence of phosphotyrosine in Cx43 immunoprecipitates from pervanadate-treated cells was shown by a phosphotyrosine-specific antibody and a phosphotyrosine-specific phosphatase. Pervanadate-induced Cx43 tyrosine phosphorylation was further verified by phosphoamino acid analysis, while no phosphotyrosine was present in control cells. This is the first observation of tyrosine phosphorylation of connexins in normal cells.

[Chemoprevention of cancer. Prevention of cancer in groups with increased risk]. / Kjemoprevensjon av kreft. Forebygging av kreft i grupper med forhøyet risiko.

The term chemoprevention is defined as the use of specific natural or synthetic chemical agents to reverse, suppress or prevent carcinogenic development to a tumour. Many potential chemopreventive substances are pharmacologic agents which may already be in use or are naturally occurring compounds. Short-term tests and animal models are available for identifying potential chemopreventive agents. There are several similarities between clinical chemopreventive trials and cancer prevention by dietary intervention. The cost and length of chemopreventive trials can be reduced by using validated biomarkers as endpoint instead of cancer. Experience from chemoprevention of cancer is limited, seen in relation to use of chemical agents to reduce cardiovascular disease. However, a number of international clinical trials are now going on to evaluate the use of pharmacological substances as potential chemopreventive agents.