Epidemiological studies suggest an association between chronic inflammation and increased risk for cancer, although the mechanism underlying this relationship is unresolved. In the present study, we test the hypothesis that DNA damage is induced in the epithelium of tissues during such inflammation by products of activated inflammatory cells. Individuals on long-term indwelling urinary catheterization were used as a study population. These individuals have chronic bladder inflammation and, as a population, an increased risk for bladder cancer. Urine of 29 patients and 26 age-matched non-catheterized controls was collected and micronucleus (MN) frequencies were determined in exfoliated urothelial cells in the urinary sediments. The urine from the catheterized group had large numbers of white blood cells (mean count, 26.6 +/- 3.6 cells per high-power field), indicating the presence of a chronic bladder infection and an inflammatory reaction. In contrast, white blood cells were not present in urine from individuals in the control group. There was no significant difference in MN frequencies in the 2 groups (mean frequencies, controls: 0.098 +/- 0.030%; catheterized: 0.140 +/- 0.025%, p = 0.13). These data imply that chromosomal damage does not always occur during chronic inflammation. Although the reasons for this observation are yet to be determined, possible explanations include the pathophysiology of the inflammatory reaction and the influence of vitamins, non-steroidal anti-inflammatory drugs and the catheter itself in protection against inflammatory cell-mediated DNA damage.

The objective of this study was to determine whether supplementation of the diet with beta-carotene has a protective effect against fetal growth retardation of rats exposed to cigarette smoke during gestation. Pregnant rats were assigned to four treatment groups. Group 1 (beta-carotene plus smoke) was fed ad libitum lab chow supplemented with 0.2% of beta-carotene and exposed to cigarette smoke daily for a 2-hour period throughout gestation. The second group (placebo plus smoke) was exposed to cigarette smoke but not supplemented with beta-carotene. The third group (pair-fed) was given amounts of unsupplemented lab chow consumed by the first group, and group four (ad libitum controls) had free access to unsupplemented lab chow. The fetuses of rats exposed to cigarette smoke weighed significantly less than those from the pair-fed and ad libitum control groups. However, the fetal weights of the beta-carotene plus smoke and the placebo plus smoke groups were similar. These findings indicate that supplementation of lab chow diet with high levels of beta-carotene does not prevent retardation of fetal growth due to in utero cigarette smoke exposure.

To evaluate the effect of chronic maternal alcohol consumption on liver vitamin A levels in both fetus and mother, female Sprague-Dawley rats were divided into three groups: alcohol, pair-fed and control. The alcohol group was given lab chow ad libitum and ethanol in drinking water for 4 weeks prior to breeding (10% the first week and 20% during the next 3 weeks) and 30% ethanol from day 1 to 21 of gestation. The pair-fed group received the same amount of lab chow consumed by the alcohol group plus corn starch calorically equivalent to the ethanol consumed. The control group was given lab chow ad libitum. on day 21 of gestation the fetuses of dams consuming alcohol were significantly smaller than those of the pair-fed and control groups. Maternal liver vitamin A concentrations (mu-mol/g) were similar in all three groups, while the amount of vitamin A present in the total liver was higher in the control group when compared with the alcohol and pair-fed groups. In the fetal livers, the concentration of vitamin A was higher in the alcohol fetuses than in the other two groups. It is concluded that chronic alcohol consumption before and during pregnancy leads to an increase in fetal liver vitamin A concentration but has no effect on maternal vitamin A levels when compared with pair-fed controls.

To determine the effect of chronic ethanol ingestion on the absorption of vitamin A, adult male rats were given 10% ethanol in drinking water for one week, 20% for the next two weeks and 30% for the last three weeks plus rat chow ad libitum. Pair-fed controls received the same amount of rat chow as that consumed by the ethanol group, but the ethanol was substituted isocalorically with starch. Ad libitum controls received rat chow only. After six weeks on these diets the animals were given 750 μg of vitamin A by stomach tube and blood was obtained from the tail at 0, 1, 2, and 3 hours after administration of the vitamin. There were no significant differences in plasma vitamin A levels among the three animal groups at time 0 (Alcohol, 17.6 μg/100 ml; pair-fed, 25.8; control, 28.3). At 1 hour, the maximum mean plasma vitamin A rise was significantly lower in the alcohol group, (47.5 μg/100 ml) than in either the pair-fed (105.5) or the control group (105.6). The findings were similar at the 2 hour interval. At 3 hours the vitamin A levels declined substantially and were no longer significantly different among the three groups. The hepatic vitamin A levels were significantly lower in the alcohol group than in the ad libitum controls. Compared to the pair-fed controls, the liver vitamin A levels were not significantly lower in the alcohol group. These findings indicate that chronic alcohol consumption by rats reduces the rate of vitamin A absorption from a test dose administered intragastrically but does not significantly alter liver vitamin A stores when compared with pair-fed controls.

It has been previously shown that plasma vitamin A concentration was decreased in both diabetic patients and streptozotocin diabetic rats. In the present study the effect of insulin administration to streptozotocin diabetic rats on plasma and liver vitamin A levels was determined. The daily insulin treatment began 3 weeks after the animals had been made diabetic and continued for 3 weeks. Plasma vitamin A levels were significantly lower in the diabetic rats than in the nondiabetic controls. In the insulin-treated diabetic rats, the plasma vitamin A together with the plasma glucose reverted to the control levels. Livers from the insulin-treated diabetic animals stored vitamin A in about the same amounts as did the livers of the untreated group but significantly more than those of the nondiabetic controls. The liver stores of vitamin A seem to reflect the amount of vitamin A consumed. These results demonstrate that treatment of diabetic rats with insulin restores plasma vitamin A to normal levels. However, the mechanism by which it does so remains to be determined.

To investigate the feasibility of measuring DNA‐carcinogen adducts in the lungs of non‐surgical patients, endobronchial biopsies were obtained from 78 patients undergoing routine diagnostic bronchoscopy. Lung cancer was present in 37 (47%) of the patients. DNA was isolated from the tissues and analyzed by HPLC‐ or nuclease‐PI ‐enriched 32P‐postlabelling, using procedures selective for aromatic adducts. Chromatograms from all 28 current smokers showed a distinctive diagonal adduct zone which was present in only 24 of 40 ex‐smokers and 4 of 10 lifetime non‐smokers. Adduct levels and chromatographic patterns were similar in bronchial tissue from different lobes of the lung, in bronchial and alveolar tissue, and in tumor and non‐tumor bronchial tissue taken from the same subject. Bronchial DNA adduct levels were strongly associated with cigarette smoking status and dropped rapidly after smoking ceased. Higher levels of DNA adducts seen in the lung‐cancer patients were mainly due to cigarette smoking. Frequent alcohol intake was the only dietary factor associated with higher levels of bronchial DNA adducts. We conclude that the level of bronchial DNA adducts is strongly associated with cigarette‐smoking history and with alcohol intake, but is not associated with lung cancer independently from its relation to smoking. The results indicate the feasibility of using 32P‐postlabelling to detect and quantitate genetic damage in bronchial biopsy specimens.

In order to investigate the putative association between chemical contamination in western Lake Ontario and high prevalences of fish tumors, sediments from Hamilton Harbour and Oakville Creek in Lake Ontario and reference sites in non-polluted areas of Ontario, Canada were collected and extracted for organic contaminants. Sediment extracts from Hamilton Harbour had the highest concentrations of polychlorinated biphenyls and organochlorine insecticides (ppb) and contained very high concentrations of polynuclear aromatic hydrocarbons (ppm); although the levels of these compounds varied widely with sampling location in the harbor. A sediment extract from Hamilton Harbour was mutagenic in the Ames bacterial assay, both with and without microsomal activation. High levels of aromatic DNA adducts were induced in cultured mouse C3H1OT1/2 cells after in vitro exposure to Hamilton Harbour sediment extract. In two separate carcinogenicity experiments involving a sac fry microinjection assay with rainbow trout (Oncorhynchus mykiss), Hamilton Harbour sediment extract induced hepatocellular carcinomas in fish. No hepatic neoplasms were observed in fish that had been treated with sediment extract from Oakville Creek, or with extract from a reference sediment. The significance of these results is discussed in relation to the distribution of neoplasms in feral fish within western Lake Ontario.

Genotoxicity in the urine of orchardists occupationally exposed to pesticides was investigated. Urine samples were obtained during pre-spraying and spraying periods from 22 non-smoking orchardists who spray large amounts of pesticides during the fruit growing season. For comparison purposes, urine was collected from 11 non-smoking personnel at an agricultural research station located near the application site and from 21 non-smoking individuals (reference controls) in a non-agricultural area. Organic material was isolated from urine by preparative reversed-phase high-pressure liquid chromatography, and assayed for clastogenic activity using Chinese hamster ovary cells. Urine samples collected during the pre-spraying period showed no significant differences in clastogenic activity compared to that found for the reference control group. However, clastogenic activity of urine specimens collected during the spraying period was significantly elevated (p < 0.001) for the highly-exposed orchardists, but not for the research station personnel. Clastogenicity of orchardists' urine was observed within 8 h of pesticide application.

We have recently assessed the feasibility of using two approaches for biological monitoring of pesticide-exposed individuals: (a) the detection of chromosome-damaging components in urine specimens, and (b) the analysis of urothelial cells for the presence of chromosomal damage, as measured by the frequency of micronuclei. Urine specimens were collected from non-smoking orchardists in the Okanagan Valley, B.C. during the pre-spraying and pesticide spraying periods of the fruit growing season. The urine was concentrated by preparative reversed-phase high-pressure liquid chromatography, and assayed for chromosome-damaging activity using Chinese hamster ovary cell cultures. A significant elevation in chromosome-damaging activity was observed only in the urine collected during the spraying period. Samples from the same individuals obtained during the pre-spraying period did not differ from non-smoking controls. Urothelial cells collected by centrifugation of urine from a single void proved to be insufficient in number for analysis of micronuclei. This approach may be applicable if multiple urine samples are obtained. A brief description of our experience with micronuclei analysis in urothelial cells from individuals in other populations is also presented.

The sensitivity of 32P-postlabeling analysis for the quantitation of DNA-carcinogen adducts can be sharply increased by enriching adducted nucleotides relative to normal nucleotides in digested DNA samples prior to postlabeling. Normal and adducted nucleotides from benzo[a]pyrene-adducted DNA were injected onto a reverse phase HPLC column. Normal nucleotides were eluted with 5% methanol, 95% 1 M ammonium formate, pH 3.5. Hydrophobic adducted nucleotides were then recovered from the column using either a linear gradient of methanol (to fractionate adducts) or a step gradient of methanol to elute all hydrophobic adducts in one fraction. Recovered chromatographic fractions were dried, postlabeled with 32P, and aromatic DNA--carcinogen adducts were detected and quantitated using TLC on polyethyleneimine cellulose. Results from the HPLC adduct enrichment procedure were generally similar to those from an alternative enrichment scheme utilizing nuclease P1 to selectively dephosphorylate normal nucleotides. HPLC and nuclease P1 procedures are easily combined for the dual analysis of DNA samples from organisms exposed to environmental carcinogens.

Internal dosimeters that can provide information about responses to chemopreventive agents in a short time would be invaluable for planning treatment protocols for large-scale intervention trials. Micronuclei meet many of the prerequisites of a good intermediate endpoint. They can be quantified in cultured cells, animal tissues and human exfoliated cells and biopsies. With image scanning, up to 10(5) cells can be screened for micronuclei within a few minutes. The predictive value of micronuclei has been demonstrated using cultured cells exposed to carcinogens and chemopreventive agents and using oral mucosa of betel-quid chewers. DNA adducts, as detected by 32P-postlabelling techniques, could conceivably be another potentially useful marker. However, prior to their use in intervention trials, interindividual variations in their levels in primary, secondary and nontarget tissues and the relationship with doses of carcinogens must be established. The wide scatter of DNA adduct levels in the bronchial mucosa of smokers and of nonsmokers reveals one difficulty that can be encountered using this marker in intervention trials.

Beta-carotene levels of exfoliated oral mucosa cells can be increased severalfold by the oral administration of this provitamin. Beta-carotene was estimated by HPLC analysis in pronase-treated exfoliated cells obtained by brushing the entire oral mucosa with a toothbrush. A small percentage of individuals did not respond with an increase of beta-carotene in their mucosa cell in spite of a relatively large intake of the provitamin (360 mg in 4 days, or 2880 mg in 16 weeks, respectively). Levels of beta-carotene in the mucosa cells are affected by the concurrent administration of vitamin A: 0.27 ng beta-carotene per 106 cells in the placebo group, 1.79 ng following the intake of beta-carotene (180 mg/week for 16 weeks), and 4.29 ng after beta-carotene (180 mg/week for 16 weeks) plus vitamin A (100,000 IU/week for 16 weeks) consumption. The considerable variations in tissue levels of beta-carotene following its oral administration must be taken into account when cancer intervention trials using this agent are designed and evaluated.

Exfoliated mucosal cells were collected from the oral cavity of three groups at high risk for oral cancer: Indian betel nut chewers, Filipino inverted smokers (burning end of cigar in mouth) and Indian Khaini tobacco chewers. DNA was extracted from these samples, as well as from samples of exfoliated cells of Canadian non-smoking controls. DNA was analyzed for the presence of aromatic DNA adducts using 32P-postlabelling analysis. Five chromatographically distinct adducts were found in samples from both the high risk groups and the nonsmoking controls. Individual adducts were detectable in approximately 30-95% of samples, depending on the adduct and population group. Estimated levels of specific adducts ranged from non-detectable (prevalence relative to normal nucleotides less than 1 X 10(-9)) to occasionally greater than 1 X 10(-7). No adducts were found in high risk groups which did not also appear in control subjects.

The usefulness of an in vitro test system to predict the inhibitory effect of beta‐carotene on the genotoxic activity of carcinogens/mutagens was explored. To facilitate the comparison of data obtained from cultured cells (CHO) and from exfoliated human cells, end‐points were used which can be quantitated in both cell systems: the frequency of micronuclei for estimating the effect of genotoxic agents, and cellular levels of beta‐carotene as a protective agent. In CHO cells, beta‐carotene inhibited the clastogenic and micronu‐cleus‐forming effect of methyl methanesulfonate (MMS) and 4‐nitroquinoline‐l‐oxide (4NQO), but had no protective action against gallic acid, tannic acid, an aqueous extract of areca nut or H 2 O 2 . The extent of inhibition depended on the ratio of beta‐carotene to MMS. Doses of beta‐carotene which exerted a protective effect in vitro ranged from approximately 2 to 5 ng per 10 6 CHO cells. Comparable levels of beta‐carotene were previously found to reduce the frequency of micronucleated exfoliated cells from the buccal mu‐cosa of tobacco and areca‐nut chewers (Stich et al. , 1984 b ).

Organic material from the urine of smokers, coffee drinkers, and controls was extracted and separated into 3 fractions of differing hydrophobicity using preparative reversed-phase high-pressure liquid chromatography. Fractions were assayed for clastogenic activity using Chinese hamster ovary cells. Smoking, coffee drinking, or both habits together resulted in a substantial increase in the genotoxicity of organic material in all 3 fractions. The clastogenicity of fractions 1 and 2 (the two most hydrophilic) was abolished by the addition of either catalase or superoxide dismutase to the Chinese hamster ovary cell system, suggesting the involvement of active oxygen species in the clastogenic response. Clastogenicity of fraction 3, however, was resistant to the action of catalase and superoxide dismutase. Fractions were tested for their ability to generate hydrogen peroxide in vitro during a 10-min incubation at elevated pH. Fractions 2 and 3, but not fraction 1 from smokers, coffee drinkers, or those with both habits generated significantly more hydrogen peroxide at high pH than did the corresponding fractions from control subjects. For fractions 2 and 3 but not 1, the ability of a sample to generate hydrogen peroxide at high pH was positively correlated with its ability to generate chromosome aberrations at neutral pH in tissue culture. The data indicate that both coffee drinking and cigarette smoking result in the appearance of clastogenic materials in urine, and suggest that such clastogenic agents may produce chromosome aberrations via the production of active oxygen species.

Small columns of XAD-2 resin have been widely used to extract and concentrate mutagenic materials from urine. Using analytical HPLC and assays for clastogenicity with Chinese hamster ovary cells, we found that small columns of XAD-2 resin (1.5 ml bed volume) retain only a small percentage of organic material and undetectable amounts of genotoxic activity in urine samples. Increasing the size of the XAD resin bed resulted in better recoveries, but much organic material was still lost by overloading of the column. In contrast, when urine was acidified and chromatographed by preparative reversed-phase HPLC using large-bed-volume (500 ml) commercial columns, retention of hydrophobic organic material from urine was excellent. Subsequent stepwise elution of the column with increasing concentrations of acetone produce 3 fractions of organic material of increasing hydrophobicity. When urine from smokers was analysed, all 3 fractions contained material which was clastogenic to Chinese hamster ovary cells. The procedures developed are suggested as a new general purpose approach to the isolation of genotoxic materials from urine.

The frequency of exfoliated cells with micronuclei (MNC) was used to estimate the genotoxic effect of smokeless tobacco (snuff) on the oral mucosa and to follow the response to the administration of beta‐carotene (180 mg/week, given twice weekly in 6 capsules of 30 mg each). The pilot trial was carried out with Inuits in Gjoa Haven, Northwest Territories, Canada. Their traditional diet, which is rich in caribou and seal meat and liver but low in vegetables and fruits, leads to “normal” serum levels of retinol (447 ng/ml in non‐users of tobacco and 463 ng/ml in tobacco users) but low levels of beta‐carotene (57 ng/ml for non‐users of tobacco and 47 ng/ml for users). Prior to the twice‐weekly administration of beta‐carotene, the frequency of MNC was 1.87% ± 0.92 (n =23) in the mucosa of the lower gingival groove where the tobacco was usually kept. It decreased significantly (P<0.001) to 0.74% ± 0.42 following the 10‐week oral administration of beta‐carotene capsules. The frequency of MNC did not change significantly in the group receiving a placebo and in snuff users who received no treatment over the 10‐week trial period. The size and morphological appearance of the typical snuff‐related, whitish, wrinkled patches of the mucosa where the tobacco was kept was not affected by the 10‐week treatment with beta‐carotene. Similarly, no reduction was observed in the frequency of anucleated, exfoliated mucosa cells. Beta‐carotene appears to be an efficient inhibitor of MNC in the oral mucosa of snuff users who do not suffer from any vitamin A deficiency and who have “normal” levels of retinol.

Polyphenolic compounds (PPC) isolated from betel nuts and some dietary PPC were examined for their modifying effects on N-nitrosation in vitro and in vivo. The formation of N-nitrosodiethylamine (NDEA) and N-nitrosoproline (NPRO) was either enhanced or inhibited by PPC from betel nuts, depending on (1) the structure of the PPC, (2) the pH of the reaction medium, (3) the relative concentrations of nitrite and PPC, and (4) the nature of the nitrosatable amino compounds. Both catalysis and inhibition of endogenous nitrosation of proline were observed in rats, although to a lesser extent than in vitro. Caffeic and ferulic acids, as well as the PPC-containing beverages tea and coffee, exerted inhibitory effects on endogenous formation of NPRO in two human subjects. These results demonstrate that PPC can modify the yield of endogenously formed N-nitroso compounds, and may thus effect the carcinogen burden in man.

The effect of dietary components on the levels of nitrosoproline ( NPRO ) excreted over a 24 h period in the urine was examined in volunteers ingesting known amounts of various food products. The ingestion of nitrite-preserved meats (85-170 g per meal), including canned, rolled or Yunnan ham, cured pork, luncheon meat, and various Chinese and European-style sausages, led to urinary NPRO excretion levels ranging from 2.5 to 78.5 micrograms/24 h, whereas the consumption of non-preserved meat and fish products, including chicken, herring, salmon, shrimp, ground beef (hamburger), pork chops and beef liver, led to relatively low NPRO excretion levels, ranging from 0.0 to 0.8 micrograms/24 h. The urinary NPRO levels of 22 vegetarians and 14 lacto-vegetarians averaged 0.8 and 1.4 micrograms/24 h, respectively. A change from a nitrite-preserved meat diet to a vegetarian diet was accompanied by an approximately six-fold reduction in urinary NPRO levels; however, these remained above control levels for at least 3 days following the dietary change. The relatively high NPRO levels following the ingestion of nitrite-preserved meats could not be reduced by nitrite-trapping chemicals, including ascorbic acid, ferulic acid, caffeic acid, or phenolic-containing mixtures such as coffee and tea, which were effective in suppressing endogenous NPRO formation following the intake of nitrate and proline. The high urinary NPRO levels after ingestion of preserved meat products appear to be due to the consumption of preformed NPRO . An understanding of the relative contribution of preformed and endogenously formed nitrosamines appears to be essential when designing dietary intervention programmes.