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.

Humoral antitumor immune responses in patients with breast cancer measured with the leukocyte adherence inhibition technique.

A modification of the hemacytometer leukocyte adherence inhibition (LAI) test was described. In this modification, 0.25% serum from patients with breast cancer was added with the relevant antigen to the assay system with the use of trypsinized leukocytes from control persons as indicator cells. The modified assay measured a humoral immune response. In studies of patients with untreated breast cancer (stages I and II) with the use of a KCl extract from a breast carcinoma or from MCF-7 cells as antigens, the modified LAI test was found to be at least as sensitive as was the ordinary test. In a blind study on sera collected from patients with breast cancer 0.5-2 years before the LAI measurements and stored at -20 degrees C, 15 of 18 (83%) patients had a positive response. Whereas the ordinary LAI test is limited to the use of fresh blood, the present test can be performed with small amounts of serum that can be frozen and stored.

Removal of inhibitors against RNA-directed DNA polymerase activity in human milk.

Milk from a number of species (e.g., man, mouse, rat, dog, and cow) contains inhibitors of the RNA-directed DNA polymerase. When attempts are made to isolate virions from the milk, part of the inhibitors follow the virions in the purification. The amount of inhibitors varies in different milk samples. These inhibitors can probably account for the large discrepancies reported in studies of the presence of oncornaviruses in human milk. Phosphatases bound to subcellular particles or fragments seem to be the most important inhibitors in the milk interfering with the RNA-directed DNA polymerase assay. It is shown that the inhibitory enzymes can be completely removed by sedimentation of the milk through a Metrizamide gradient.

Assay for initiators and promoters of carcinogenesis based on attachment-independent survival of cells in aggregates.

A cell line (HRRT) derived from a hereditary renal rat tumor has been used in an assay for initiators and promoters of carcinogenesis based on attachment-independent survival in aggregates. Treatment with single noncytotoxic doses of the carcinogens urethan (1 mM), N-methyl-N-nitrosourea (30 microM), and benzo(a)pyrene (0.2 microM) for 1 hr did not affect survival of HRRT cells in the aggregate assay system. However, when carcinogen treatment was followed by exposure of the cells to the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (0.16 microM), a considerable increase in survival was observed. With urethan as an initiator, it was found that tumor promoters (12-O-tetradecanoylphorbol-13-acetate and phorbol-12, 13-didecanoate) induced a considerable response in the assay system, while nonpromoting phorbol esters (4-O-methyl-12-O-tetradecanoylphorbol-13-acetate and 4 alpha-phorbol-12, 13-didecanoate) did not affect the survival. Exposure of HRRT cells to NiSO4 (40 microM) for 3 hr did not influence cell survival in the aggregate form. However, subsequent treatment of the cells with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate induced a marked increase in the number of viable cells. Moreover, treatment of HRRT cells with a nontransforming dose of urethan (1 mM) for 1 hr followed by continuous exposure to nickel sulfate (40 microM) also increased cell survival in the aggregate form. These results support the view that nickel sulfate may act as both an initiator and a promoter in mammalian cell transformation. The present data also indicate that the aggregation assay system using the HRRT cell line may be a valuable in vitro screening assay for putative initiators and promoters of carcinogenesis.

Metal salts as promoters of in vitro morphological transformation of hamster embryo cells initiated by benzo(a)pyrene.

The hamster embryo cell bioassay has been used to study the effect of metal salts on morphological transformation. A synergistic enhancement of the transformation frequency was found for the combined treatment with organic carcinogens [benzo(a)pyrene, N-hydroxy-2-acetylaminofluorene, and 4-nitroquinoline 1-oxide] and nickel sulfate, cadmium acetate, or potassium chromate. Chromic chloride and zinc chloride did not induce transformation themselves, and they had no effect on the transformation frequency when tested in combination with benzo(a)pyrene. The synergistic effect between benzo(a)pyrene and nickel sulfate or cadmium acetate was also apparent when the cells were treated sequentially with the chemicals. When the cells were first exposed to benzo(a)pyrene, both nickel sulfate and cadmium acetate showed a promotion-like effect similar to that obtained with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Moreover, when 12-O-tetradecanoylphorbol-13-acetate or benzo(a)pyrene were used as promoting agents, both nickel sulfate and cadmium acetate were able to initiate morphological transformation. The data suggest that the metal salts are more potent as promoters than they are as initiators. The present findings may be of importance in relation to carcinogenicity of metal compounds to humans.

Cellular antitumor immune response in women with risk factors for breast cancer.

The leukocyte adherence inhibition technique was used to study cell-mediated immunoactivity to breast adenocarcinoma. In a group of 74 patients with untreated breast cancer in Stages I and II, 69% showed a positive response, while 48% of the 25 patients in Stages III and IV had reactive leukocytes. Among 43 control persons, only 2 women showed positive responses. In a group of 161 women with benign breast diseases, 24% showed a positive reaction against breast carcinoma extract. The percentage of positive responses was higher than the average of women with benign disease among those who had a mother or a sister with breast cancer, who had previously had benign breast lumps removed, who had microcalcifications in their breasts, or for whom an increased intraductal epithelial proliferation was found in their biopsies. Twenty-eight women with benign breast diseases and two or more of the above factors were assigned to the high-risk group, in which 43% had reactive leukocytes. Of the 50 women with one risk factor, 34% showed a positive response, while of the 83 women in the low-risk group, with none of the above factors, only 11% had a positive reaction. The results suggest that the leukocyte adherence inhibition test may be used to identify groups of women who have a high risk for breast cancer.

Role of ions in the regulation of porcine lactate dehydrogenase.

Different ions affect the H4 and M4 isoenzymes of porcine lactate dehydrogenase (L-lactate: NAD+ oxidoreductase, EC 1.1.1.27) in the same way, inhibiting the enzyme at low pyruvate concentrations, whereas at high pyruvate concentrations, the activities were enhanced. The inhibition was competitive with regard to pyruvate and NADH. The enhancement of the enzyme activity at high pyruvate concentration is due to the increase in the Km value for pyruvate, implying that higher substrate concentrations are needed to obtain substrate inhibition. Sulphate behaved differently from the other ions. It inhibited in a noncompetitive manner with regard to pyruvate and did not activate the enzyme at high pryvuate concentration. The effect of ions increased with the size of the anion. The ionic strength was of less importance.

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.

Synergistic effect on morphological transformation of hamster embryo cells by nickel sulphate and benz[a]pyrene.

Morphological transformation and induction of somatic mutation in the hamster embryo cell bioassay have been used to study whether the carcinogenicity of nickel is affected by polycyclic hydrocarbons. The transformation frequency was found to increase with increasing concentration of nickel sulphate, benz[a]pyrene (BP) and methylcholanthrene. In experiments with combinations of nickel sulphate and BP, the transformation frequencies used for all concentrations were higher than for compounds tested separately. The greatest enhancement was found using 5 micrograms/ml NiSO4 . 6H2O and 0.78 microgram/ml BP. The transformation frequency obtained with this combination was 10.7%, compared to 0.5% and 0.6% for the individual substances. No synergistic effect could be detected between nickel sulphate and methylcholanthrene (MC). In experiments measuring somatic mutation by selection for ouabain resistance, the mutation frequency was likewise found to be significantly higher than expected in mixtures of nickel sulphate and BP. The present demonstration of the synergistic effect between nickel sulphate and BP is of interest with the potentiating effect of cigarette smoking on development of lung cancer among nickel refinery workers.

Potentiating effect of cigarette smoke extract on morphological transformation of hamster embryo cells by benzo[alpha]pyrene.

Morphological transformation in the hamster embryo cell bioassay has been used to study the possibility that carcinogenicity of benzo[alpha]pyrene (BP) is affected by cigarette smoke extract and whether smoke extract will promote transformations initiated by BP. The transformation frequency increases wth increasing concentrations of BP and smoke extract. In experiments with a combined treatment of BP and smoke extract, the transformation rates were higher than expected for all concentrations from experiments with the compounds tested separately. The greatest potentiating effect was found using 0.01 micrograms/ml BP and 1 microgram/ml smoke extract. The transformation frequency obtained with this combination was 4.3% compared to 1.4% and zero respectively for the individual substances. In experiments where cells were treated sequentially with BP (0.05 micrograms/ml) for 4 days followed by smoke extract (1 or 5 micrograms/ml) for the next 4 days, the transformation frequency was significantly higher than expected on the basis of the compounds tested separately. The demonstration of a synergistic effect between BP and cigarette smoke and the promotion-like effect of smoke extract on BP-initiated transformations of hamster embryo cells are of interest in relation to the higher frequency of lung cancer found in areas with high air pollution compared to rural areas.

Promotional effect of different phorbol esters on morphological transformation of hamster embryo cells.

Hamster embryo cells were sequentially exposed to benzo[a]pyrene (BaP) (3 days) and different phorbol esters (4 days). Compounds which have been shown to act as tumor promoters in mouse skin carcinogenesis, showed a promotion-like effect on the formation of morphologically transformed colonies, while non-promoting analogs did not enhance the transformation frequency. In experiments where the exposure sequence was reversed, i.e. 12-O-tetradecanoyl phorbol-13-acetate (TPA) in the first exposure period and BaP in the second period, no significant enhancement of the transformation frequency was observed. The exposure to a low concentration of BaP in the second exposure period following a higher concentration in the first period, resulted in a strong enhancement of the transformation frequency suggesting that BaP also acts as a promoter.

Caffeine and other phosphodiesterase inhibitors are potent inhibitors of the promotional effect of TPA on morphological transformation of hamster embryo cells.

The phosphodiesterase inhibitors caffeine, theophylline, aminophylline and isobutyl-methylxanthine (IBMX) were found to inhibit induction of morphologically transformed hamster embryo cell colonies by sequential exposure to benzo[a]pyrene (BaP) and the tumor promoter TPA. Almost complete inhibition of cell transformation was observed when 50 micrograms/ml theophylline, aminophylline, IBMX, or 200 micrograms/ml caffeine was present together with the tumor promoter. The compounds had no effect on the transformation frequency when present together with the initiator, BaP, in the first exposure period. Substances that stimulate the adenylate cyclase and the addition of exogenous dibutyryl-cAMP had similar inhibitory effects.

Role of circulating antibodies in the humoral leukocyte adherence inhibition response of lung and breast cancer patients.

A modified leukocyte adherence inhibition (H-LAI) assay has recently been developed in which 0.25% serum from the patient is added to the assay system in combination with the relevant antigen. Trypsinized leukocytes from control persons are used as indicator cells. In the present work, the nature of the humoral factor in serum from breast and lung cancer patients is studied. 3.5 M KCl extracts from the cell lines MCF-7 and Calu-1 were used as breast and lung cancer antigen, respectively. It was found that the humoral factor involved in the H-LAI response was precipitated from the sera by addition of ammonium sulphate to 50% saturation. This factor could be removed by passage through an affinity column with the relevant antigen bound to the matrix. Stable complexes were formed between the humoral factor and the relevant antigen, and could be precipitated by polyethylene glycol. When different anti-immunoglobulins were added to the sera, the humoral factor was specifically removed by addition of anti-IgG antibodies. The data presented indicate that the humoral factor in sera from patients with breast and lung cancer is antitumor antibodies of IgG nature.

Immune responses in lung cancer patients measured by a modified leukocyte adherence inhibition test using serum.

A modified leukocyte adherence inhibition (H-LAI) assay was used to study immunological factors in serum from lung cancer patients. In this test, 0.25% serum was added to the assay system, together with tumor antigen and trypsinized leukocytes from control persons. Extracts from a human lung cancer cell line (Calu-1) and a human breast cancer cell line (MCF-7) were used as antigens. The results obtained were compared with data found with the original hemocytometer (C-LAI) assay. Of 21 lung cancer patients studied, 20 (95%) gave a positive response in both the H-LAI and the C-LAI assay systems against Calu-1 antigen. Only 1 of the patients gave a positive response in the H-LAI system against MCF-7 antigen, while 3 patients (14%) responded in the C-LAI assay. None of the 14 control persons tested gave a positive response. While the C-LAI assay was limited to the use of fresh blood, the H-LAI system was performed on small amounts of serum. The serum could be stored in the frozen state for a long time period. The results indicate that the H-LAI assay possesses at least the same sensitivity and specificity as the original C-LAI test.

Effects of hepatic peroxisome proliferators and 12-O-tetradecanoyl phorbol-13-acetate on catalase and other enzyme activities of embryonic cells in vitro.

The effects of the hepatic peroxisome proliferators (HPPs) clofibrate, di-(2-ethylhexyl)-phthalate (DEHP), mono-(2-ethylhexyl)phthalate (MEHP) and 2,4-dichlorophenoxy acetic acid (2,4-D) on the activities of some peroxisome-associated enzymes and marker enzymes for other organelles, have been studied in primary Syrian hamster embryo (SHE) cells and Wistar rat embryo (WRE) cells. The majority of the cells are fibroblast-like. 12-O-Tetradecanoyl phorbol-13-acetate (TPA) was included as it has been suggested that it may act as a peroxisome proliferator. The specific activities of catalase, fatty acyl-CoA oxidase (FAO) and peroxisomal beta-oxidation were approximately 100-fold lower in the embryonic cells than in rat hepatocytes. Other peroxisome-associated oxidases were not detected. The dihydroxyacetone-phosphate acyltransferase (DHAPAT) activity was comparable to that in rat liver. Marker enzymes for other organelles had specific activities comparable to rat hepatocytes. Catalase was shown by digitonin titration to be contained in a peroxisome-like compartment in both SHE and WRE cells. Clofibrate, DEHP and MEHP increased the catalase activity, which might suggest peroxisome proliferation. However, the findings that FAO and peroxisomal beta-oxidation did not increase or only very slightly, argue against peroxisome proliferation. 2,4-D and TPA induced no or only a very slight increase in the catalase activity.

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.

Effect of 1,1'-(2,2,2-trichloroethylidene)-bis(4-chlorobenzene) (DDT) on gap junctional intercellular communication and morphological transformation of Syrian hamster embryo cells.

The organochlorine insecticide 1,1'-(2,2,2-trichloroethylidene)bis(4-chlorobenzene) (DDT) did not induce or promote induction of morphological transformation in Syrian hamster embryo (SHE) cells, but it was a potent inhibitor of gap junctional intercellular communication (GJIC). The kinase inhibitor staurosporine did not affect DDT induced inhibition of GJIC, although it has been shown to decrease the inhibitory effect of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on GJIC. In addition, pretreatment with TPA made the cells refractory to further TPA induced inhibition of GJIC, while they remained sensitive to DDT. Thus, DDT and TPA inhibit GJIC through different mechanisms. Elevation of cellular cyclic adenosine monophosphate (cAMP) level by exposure to forskolin counteracted the inhibitory effect of DDT similar to that observed for TPA. Continuous exposure to DDT at concentrations near the effective concentration (50%) value (EC50 value) resulted in a slight recovery of GJIC following the initial inhibition. This recovery was not accompanied by the cells becoming refractory to further DDT induced inhibition of GJIC. The recovery of GJIC after removal of the DDT containing medium seemed to be related to a reduction in the amount of cell-associated DDT.

Effect of protein kinase C inhibitors on TPA-induced inhibition of gap junctional intercellular communication and induction of transformed morphology in Syrian hamster embryo cells.

Staurosporine and H-7, but not polymyxin B, were found partly to suppress the effect of TPA on both inhibition of gap junctional intercellular communication (GJIC), down-regulation of EGF-binding and induction of transformed morphology in Syrian hamster embryo (SHE) cells. The parallel effect of these kinase inhibitors suggests that TPA induces the effects through activation of protein kinase C (PKC). This is consistent with the view that PKC is involved in both the induction of transformed morphology and the inhibition of GJIC. All the inhibitors studied reduced PKC activity in SHE cell extracts. IC(50) values were determined to be 0.008 mum for staurosporine, 55 mum for H-7 and 16 mum for polymyxin B. The values for staurosporine and H-7 corresponded to those concentrations that suppressed TPA-induced cellular responses. The lack of effect of polymyxin B on whole SHE cells indicates that this compound does not enter the cells.

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.

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.