Paul L. Skipper

ANALYSIS OF NITRATE, NITRITE AND 15N NITRATE IN BIOLOGICAL FLUIDS

Abstract A new automated system for the analysis of nitrate via reduction with a high-pressure cadmium column is described. Samples of urine, saliva, deproteinized plasma, gastric juice, and milk can be analyzed for nitrate, nitrite, or both with a lower limit of detection of 1.0 nmol NO 3 − or NO 2 − /ml. The system allows quantitative reduction of nitrate and automatically eliminates interference from other compounds normally present in urine and other biological fluids. Analysis rate is 30 samples per hour, with preparation for most samples limited to simple dilution with distilled water. The application of gas chromatography/mass spectrometry for the analysis of 15 NO 3 − in urine after derivatization to 15 NO 2 -benzene is also described.

Elevated blood levels of carcinogens in passive smokers.

The hypothesis that involuntary exposure to tobacco smoke--passive smoking--results in greater risk of cancer was assessed by measuring the levels of two known carcinogens in the blood of 57 nonsmokers with varying degrees of involuntary exposure, including six heavily exposed bartenders. The concentrations of hemoglobin adducts of 4-aminobiphenyl, a bladder carcinogen, were significantly higher in subjects with confirmed involuntary exposure (plasma cotinine concentrations between 2 and 23 ng/ml) compared with subjects with undetectable levels of cotinine. Similarly, adducts of 3-aminobiphenyl were significantly elevated in subjects with confirmed exposure. The odds of 3-aminobiphenyl adduct levels exceeding 2 pg/g of hemoglobin were 6:7 among the confirmed exposed, compared with the odds of 2:42 among subjects with undetectable cotinine (odds ratio = 18; 95 percent confidence interval = 3.3, 94). The validity of the assay was demonstrated by showing striking declines in adduct levels among quitting smokers.

Arylamine exposures and bladder cancer risk

Occupational exposure to arylamines in industrial settings was the first known cause of bladder cancer in humans. In the United States and many developed countries, these industrial dyes have been under strict government control for decades and are believed to contribute minimally to todays population burden of bladder cancer in the West. The two other recognized, and potentially substantial sources of human exposure to arylamines are cigarette smoking and use of hair dyes. This paper reviews the latest epidemiologic findings on the relationships between smoking, hair dye use and bladder cancer risk. Results support the notion that arylamines contained in cigarette smoke and permanent hair dyes are human carcinogens. Furthermore, women may experience higher bladder cancer risk than men from comparable arylamine exposure, possibly due in part to womens higher propensity for arylamine activation relative to men.

Monocyclic aromatic amines as potential human carcinogens: old is new again.

Alkylanilines are a group of chemicals whose ubiquitous presence in the environment is a result of the multitude of sources from which they originate. Exposure assessments indicate that most individuals experience lifelong exposure to these compounds. Many alkylanilines have biological activity similar to that of the carcinogenic multi-ring aromatic amines. This review provides an overview of human exposure and biological effects. It also describes recent investigations into the biochemical mechanisms of action that lead to the assessment that they are most probably more complex than those of the more extensively investigated multi-ring aromatic amines. Not only is nitrenium ion chemistry implicated in DNA damage by alkylanilines but also reactions involving quinone imines and perhaps reactive oxygen species. Recent results described here indicate that alkylanilines can be potent genotoxins for cultured mammalian cells when activated by exogenous or endogenous phase I and phase II xenobiotic-metabolizing enzymes. The nature of specific DNA damage products responsible for mutagenicity remains to be identified but evidence to date supports mechanisms of activation through obligatory N-hydroxylation as well as subsequent conjugation by sulfation and/or acetylation. A fuller understanding of the mechanisms of alkylaniline genotoxicity is expected to provide important insights into the environmental and genetic origins of one or more human cancers and may reveal a substantial role for this group of compounds as potential human chemical carcinogens.

Antibody-antigen binding in organic-solvents

We describe, for the first time, the action of antibodies in anhydrous organic solvents. It has been demonstrated that the binding of a hapten, 4-aminobiphenyl, to the immobilized monoclonal antibody 2E11 is strong and specific not only in water but also in a variety of non-aqueous media. Further, the strength of interaction between antibody and hapten has been related to the hydrophobicity of the solvent: the more hydrophobic the solvent, the weaker the protein-ligand interaction.

Carotenoids/vitamin C and smoking-related bladder cancer

Previous epidemiological studies of fruit and vegetable intake and bladder cancer risk have yielded inconsistent results, especially with respect to the role of cigarette smoking as a possible modifier of the diet‐bladder cancer association. A population‐based case‐control study was conducted in nonAsians of Los Angeles, California, which included 1,592 bladder cancer patients and an equal number of neighborhood controls matched to the index cases by sex, date of birth (within 5 years) and race between January 1, 1987 and April 30, 1996. Information on smoking, medical and medication history, and intake frequencies of food groups rich in preformed nitrosamines, vitamins A and C and various carotenoids, were collected through in‐person, structured interviews. Beginning in January 1992, all case patients and their matched control subjects were asked for a blood sample donation at the end of the in‐person interviews for measurements of 3‐ and 4‐aminobiphenyl (ABP) hemoglobin adducts, and glutathione S‐transferases M1/T1/P1 (GSTM1/T1/P1) and N‐acetyltransferase‐1 (NAT1) genotypes. Seven hundred seventy‐one (74%) case patients and 775 (79%) control subjects consented to the blood donation requests. In addition, all case patients and matched control subjects were asked to donate an overnight urine specimen following caffeine consumption for measurements of cytochrome P4501A2 (CYP1A2) and N‐acetyltransferase‐2 (NAT2) phenotypes. Urine specimens were collected from 724 (69%) case patients and 689 (70%) control subjects. After adjustment for nondietary risk factors including cigarette smoking, there were strong inverse associations between bladder cancer risk and intake of dark‐green vegetables [p value for linear trend (p) = 0.01], yellow‐orange vegetables (p = 0.01), citrus fruits/juices (p = 0.002) and tomato products (p = 0.03). In terms of nutrients, bladder cancer risk was inversely associated with intake of both total carotenoids (p = 0.004) and vitamin C (p = 0.02). There was a close correlation (r = 0.58, p = 0.0001) between intakes of total carotenoids and vitamin C in study subjects. When both nutrients were included in a multivariate logistic regression model, only total carotenoids exhibited a residual effect that was of borderline statistical significance (p = 0.07 and p = 0.40 for total carotenoids and vitamin C, respectively). Cigarette smoking was a strong modifier of the observed dietary effects; these protective effects were confined largely to ever smokers and were stronger in current than ex‐smokers. Smokers showed a statistically significant or borderline statistically significant decrease in 3‐ and 4‐aminobiphenyl (ABP)‐hemoglobin adduct level with increasing intake of carotenoids (p = 0.04 and 0.05, respectively). The protective effect of carotenoids on bladder cancer seemed to be influenced by NAT1 genotype, NAT2 phenotype and CYP1A2 phenotype; the association was mainly confined to subjects possessing the putative NAT1‐rapid, NAT2‐rapid and CYP1A2‐rapid genotype/phenotype. The carotenoid‐bladder cancer association was not affected by the GSTM1, GSTT1 and GSTP1 genotypes.

Environmental Tobacco Smoke and Bladder Cancer Risk in Never Smokers of Los Angeles County

Cigarette smoking is a major risk factor for bladder cancer and a prominent point source of 4-aminobiphenyl (4-ABP), a recognized human bladder carcinogen. 4-ABP-hemoglobin (Hb) adducts are established biomarkers of 4-ABP exposure in humans. The role of environmental tobacco smoke (ETS) in the etiology of bladder cancer is largely unknown. As part of a large population-based bladder cancer study in Los Angeles County, California, lifetime exposure to ETS was ascertained for 148 cases and 292 control subjects who had never used any tobacco products over their lifetime. 4-ABP-Hb adducts were quantitatively measured on 230 control subjects. Female lifelong nonsmokers living with two or more smokers during childhood were significantly related to risk of bladder cancer [odds ratio (OR), 3.08; 95% confidence interval (95% CI), 1.16-8.22]. During adulthood, approximately 2-fold risks were seen among women living with a spouse/domestic partner who smoked for > or =10 years or having a coworker who smoked in an indoor environment for > or =10 years. When all sources of ETS exposure were combined, a statistically significant, dose-dependent association (P for trend = 0.03) was noted in women, with the OR for the highest category of ETS exposure being 5.48 (95% CI, 1.06-28.36). Levels of 4-ABP-Hb adducts varied by ETS exposure status among female control subjects. Mean level was lowest in women never exposed to ETS (16.4 pg/g Hb) and highest in those with current ETS exposure (23.6 pg/g Hb). ETS exposure was associated with neither bladder cancer risk nor 4-ABP-Hb adduct levels in male lifelong nonsmokers. In conclusion, ETS is a risk factor for bladder cancer in women who were lifelong nonusers of any tobacco products.

Low-energy biomedical GC–AMS system for 14C and 3H detection

Abstract The use of accelerator mass spectrometry (AMS) in biomedical research will require the development of cost-effective, laboratory-sized AMS systems that can be used in conjunction with gas and liquid phase separation techniques. This paper describes a prototype GC–AMS system designed for the detection of 14C and 3H in organic samples. The entire AMS system including the injector, ion source, tandem accelerator, and high-energy analyzer is approximately 3.5 m wide, 1.5 m high and 1 m deep. Also described are methods for converting gas chromatograph (GC) effluent to gaseous CO2 for 14C-labeled compounds. A gas-fed cesium (Cs) sputter ion source converts the CO2 into C− for injection into the AMS accelerator, allowing on-line analysis of 14C-labeled biological samples with AMS.

N7-Glycidamide-Guanine DNA Adduct Formation by Orally Ingested Acrylamide in Rats: A Dose–Response Study Encompassing Human Diet-Related Exposure Levels

Acrylamide (AA) is formed during the heating of food and is classified as a genotoxic carcinogen. The margin of exposure (MOE), representing the distance between the bench mark dose associated with 10% tumor incidence in rats and the estimated average human exposure, is considered to be of concern. After ingestion, AA is converted by P450 into the genotoxic epoxide glycidamide (GA). GA forms DNA adducts, primarily at N7 of guanine (N7-GA-Gua). We performed a dose-response study with AA in female Sprague-Dawley (SD) rats. AA was given orally in a single dosage of 0.1-10 000 μg/kg bw. The formation of urinary mercapturic acids and of N7-GA-Gua DNA adducts in liver, kidney, and lung was measured 16 h after application. A mean of 37.0 ± 11.5% of a given AA dose was found as mercapturic acids (MAs) in urine. MA excretion in urine of untreated controls indicated some background exposure from endogenous AA. N7-GA-Gua adduct formation was not detectable in any organ tested at 0.1 μg AA/kg bw. At a dose of 1 μg/kg bw, adducts were found in kidney (around 1 adduct/10(8) nucleotides) and lung (below 1 adduct/10(8) nucleotides) but not in liver. At 10, respectively, 100 μg/kg bw, adducts were found in all three organs, at levels close to those found at 1 μg AA/kg, covering a range of about 1-2 adducts/10(8) nucleotides. As compared to DNA adduct levels from electrophilic genotoxic agents of various origin found in human tissues, N7-GA-Gua adduct levels within the dose range of 0.1-100 μg AA/kg bw were at the low end of this human background. We propose to take the background level of DNA lesions in humans more into consideration when doing risk assessment of food-borne genotoxic carcinogens.

Biomonitoring of heterocyclic aromatic amine metabolites in human urine

Human exposure to heterocyclic aromatic amines such as MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline) may be monitored by measuring the levels of the heterocyclic aromatic amine in urine. In order to investigate the contribution of N-oxidation to the metabolism of MeIQx in vivo, we developed a biomonitoring procedure for the analysis and quantification of the N2-glucuronide conjugate of 2-hydroxyamino-3,8-dimethylimidazo[4,5-f]quinoxaline in human urine. Subjects (n = 66) in the dietary study ingested a uniform diet of cooked meat containing known amounts of MeIQx, and urine was collected after consumption of the test meal. A method based on solid-phase extraction and immunoaffinity separation was used to isolate N2-(beta-1-glucosiduronyl)-2-hydroxyamino-3,8-dimethylimidazo++ +[4,5-f]quinoxaline and its stable isotope-labeled internal standard from urine. The isolated conjugate was converted to the deaminated product 2-hydroxy-3,8-dimethylimidazo[4,5-f]quinoxaline by treatment with acetic acid under moderate heating. 2-Hydroxy-3,8-dimethylimidazo[4,5-f]quinoxaline and the [2H3]methyl analog were derivatized to form the corresponding 3,5-bis(trifluoromethyl)benzyl ether derivatives and quantified by capillary gas chromatography-negative ion chemical ionization mass spectrometry employing selected ion monitoring procedures. The amounts of N2-(beta-1-glucosiduronyl)-2-hydroxyamino-3,8-dimethylimidazo++ +[4,5-f]quinoxaline recovered in urine collected 0-12 h after the test meal accounted for 2.2-17.1% of the ingested dose, with a median value of 9.5%. The variability in the proportion of the dose excreted among the subjects may be reflective of several factors, including interindividual variation in the enzymic activity of CYP1A2 and/or conjugation reactions of the N-hydroxylamine metabolite with N-glucuronosyltransferase(s).