Urinary Mutagenic Activity in Workers Exposed to Diesel Exhaust

ABSTRACT


We measured postshift urinary mutagenicity (mutu; n = 306 samples) on a population of railroad workers (n = 87) with a range of diesel exhaust exposures. Postshift urinary mutagenicity was determined by a sensitive microsuspension procedure using Salmonella strain TA 98 +- $9. Number of cigarettes smoked on the study day and urinary cotinine were highly correlated with postshift urinary mutagenicity. Diesel exhaust exposure was mea- sured over the work shift by constant-flow personal sampling pumps. Respirable particle concentrations were adjusted for the contribution of environmental tobacco smoke, as estimated from nicotine concentration on treated filters. 

The relative ranking of jobs by this adjusted respirable particle concentration (ARP) was correlated with relative contact the job groups have with operating diesel locomotives. After adjustment for cigarette smoking (active and passive) in multiple regressions, there was no independent association of diesel exhaust exposure, as estimated by ARP, with postshift urinary mutagenicity among smokers or nonsmokers. An important finding is the detection of "baseline" mutagenicity in most of the nonsmoking workers. Despite the use of individual measurements of diesel exhaust exposure, the absence of a significant association in this study may be due to the low levels of diesel exposure, the lack of a specific marker for diesel exhaust exposure, and/or urinary mutagenicity levels from diesel exposure below the limit of sensitivity for the mutagenicity a s s a y .

METHODS

Study Population

All subjects worked for a single railroad in the northeast United States. Sampling was done during six field visits in the winter because our previous work had shown that diesel exhaust exposure was highest during that season . Study subjects were selected, based on job category and work location, from five job categories known to provide a range of diesel exhaust exposures among employees of similar socioeconomic status (Table 1). Employees in management and administrative positions were not studied. All subjects were studied on at least two consecutive workdays. Because of the limited population size and to maximize study power by oversampling the highest diesel exposure groups, several subjects provided more than one 2-day sample set (Table 1).

Subjects were recruited after informed consent. Personal sampling pumps with filter cassettes were attached at the beginning of the work shift, and individual logs were kept of work activities, locations, and field conditions on the study days. Pumps and samples were collected at the end of the work shift, cigarettes smoked were counted, and a spot urine was obtained. The same procedure was repeated on the second day, and the subjects also completed a questionnaire on their medical history, nonworkplace exposures, diet, cigarette smoking, and other lifestyle factors. Several nonworkplace exposure variables were tested as predictors of urinary mutagenicity, including clinical conditions, dietary intake in the previous 2 days, and nonoccupational chemical exposures. A specific dietary variable of "protective food " consisted of individuals who reported ingesting cabbage, brussel sprouts, or fish in the previous 2 days.

Diesel Exhaust and Environmental Tobacco Smoke Exposure

Personal exposure to diesel exhaust and other air contaminants was measured by constant-flow personal sampling over full work shifts, which ranged from 7 to 10 hr. The sampling method has been described in detail elsewhere . Briefly, respirable particles were collected on 37-mm Teflon-coated fiber filters (Pallflex Corp, Putnam, CT) preceded by a 10-mm nylon cyclone. Particle mass was determined gravimetrically in a room maintained at controlled temperature (70 -+ 5°F) and humidity (50 -+ 10%). Air flow through the sampling train was drawn at 1.7 liters per minute (lpm), and field calibrated (+5%) with a rotameter.

A second filter downstream from the particle filter was treated with an acid, sodium bisulfate, to bind with nicotine vapor, which is alkaline. Nicotine vapor loss by this collecting system was measured by studies of environmental tobacco smoke (ETS) exposure in an environmental chamber, and was less than 1% of the nicotine collected on the treated filters . Nicotine was measured by aqueous desorption, pH adjustment, extraction into heptane and gas chromatography with nitrogen phosphorus detection. Recovery of 0.5 ~g nicotine spikes on clean filters was 98 --+2%. The limit of detection for field sampling at 1.7 lpm for 8 hr is an average nicotine concentration of 0.2 p~g/m3, based on GC limits of quantitation and linearity.

Exposure to diesel exhaust among diesel-exposed workers was estimated from respirable particle concentration (RSP) adjusted for respirable particles from ETS as estimated by the nicotine concentration on each sample . The correction factor for ETS particles from nicotine concentrations was derived from environmental chamber studies and confirmed by regressing RSP on nicotine among workers in this study who were non-diesel exposed. Both methods yielded a factor of 8.6 as the coefficient of nicotine concentration, so ETS particle concentration was determined by multiplying nicotine concentration by 8.6, This value was subtracted from individual filter RSP results to yield an " adjusted " RSP (ARP).

Mutagenicity Testing

We used a microsuspension (preincubation) procedure, previously developed to detect mutagens in the urine of cigarette smokers and non-tobacco users. The method is at least l0 times more sensitive than the standard plate incorporation test based on absolute amounts of compound added per plate . Briefly, Salmonella were grown overnight in oxoid nutrient broth, harvested by centrifugation, and resuspended in iced phosphate-buffered saline (PBS, 0.15 M, pH 7.4) to a concentration of 1 x l0 l° cells/ml. For the microsuspension assay the following ingredients were added in order to a 12 x 75-ram glass culture tube placed in an ice bath: 0.1 ml S9 , 0.005 ml urine extract in dimethyl sulfoxide (DMSO), and 0.1 ml bacteria in PBS (1 × 109 bacteria per tube).

The mixture was incubated at 37°C for 90 min with shaking. After incubation, the tubes were placed in an ice water bath and removed singly from the ice bath, and 2 ml of molten top agar containing 90 nmole of both histidine and biotin was added. The molten suspensions were mixed immediately with a Vortex mixer and poured onto minimal glucose plates. The plates were incubated at 37°C in the dark for 48 hr and counted using an automatic colony counter. Genetic markers for the strains were routinely verified. All extraction and testing were carried out in a room fitted with yellow fluorescent lights to minimize potential photooxidation.

Urine samples were collected in washed, sterile amber-colored glass or polyethylene bottles and immediately placed on ice and stored at -213°C. Urines were extracted with XAD-2, following the methods of and ). Urine extracts were tested for mutagenic activity in batches with their own PBS controls. All urine samples or PBS control samples were tested in duplicate at a minimum of three urine equivalent doses, 2.5, 5, and l0 ml, with and without $9. The mutagens 2-aminofluorene and benzo[a]pyrene served as positive controls for + $9, and 2-nitrofluorene and 4-nitroquinolone-Noxide served as positive controls for - $ 9 assays. All positive control mutagens were tested at three doses in triplicate. Four or five plates were routinely used for the DMSO standard which provided the 0.0 concentration. Only single values are presented for all replicate samples. Thus, the sample size for all samples reflects the number of specimens collected.

Mutagenicity (revertants per milliliter equivalent of urine) was determined from a straight-line least-squares regression, followed by subtraction of the slope from the extraction control PBS sample. Points starting from the highest dose were dropped until the t test for quadratic curvature was "nonsignificant" at p > 0.15. 

Urinary concentration of cotinine was determined by P. Jacob, L. Yu, and N. Benowitz, using a multistep extraction procedure followed by gas chromatographic analyses . Concentrations of urine markers were standardized for urine creatinine concentration.

Statistics

Diesel exhaust exposure was evaluated as a predictor of urinary mutagenicity in three ways: (1) by evaluation of mean urinary mutagenicity among samples from workers classified by a priori levels of diesel exposure as estimated by job categories; (2) by evaluation of urinary mutagenicity levels among ordered strata of diesel-exposed workers as estimated by ARP; and (3) by a full multiple regression model in which the effect of "diesel exposure" was expressed as an interaction between the amount of respirable particles and nicotine in the air and work at jobs with known potential for diesel exposure. Results were adjusted for other predictors of mutagenicity as determined by multiple linear regressions. To accommodate the multiple observations per person that were taken in the study, an estimated generalized least-squares program (BMDP3V) was used, which allowed for within- and between-person variance components. All analyses were done by SAS 5.16 or BMDP procedures on a VAX 11/750 computer.

RESULTS


The 87 subjects studied had a mean age of 47.2 -+ 9.7 (x -+ SD) and mean years of railroad work of 16.1 + 10.8 (Table 1). Engineers tended to be older than subjects in other job categories, which is consistent with railroad career paths.

Thirty-six percent of subjects (n = 31) were cigarette smokers. Four nonsmokers and two smokers used other forms of tobacco. This yielded 95 urine samples for cigarette smokers (32%), 10 samples for cigarette smokers who used other tobacco products (3%), 189 non-tobacco user samples (62%), and 12 samples from subjects who used other tobacco products (4%). 

Results for cigarette smokers who used other tobacco products were similar to those for cigarette smokers and, therefore, were analyzed together. Results for urine analyses of non-cigarette-smoking subjects who used other tobacco products were excluded or considered separately.

The highest rate of tobacco use was among the carmen studied (72% of subjects), with lower rates of smoking present for the other job categories (2%50% of subjects) (Table 1). Among smokers, the average number of cigarettes smoked during the day prior to urine sample collection was 14.4 -+ 8.1. For most analyses the number of cigarettes smoked during the study day up to the time of urine collection was used, since it was felt to better reflect markers present in the urine at the end of the shift than the number of cigarettes smoked only during the shift.

Some samples were missing or not analyzed from each source in this large, multidisciplinary field investigation. The reasons were different for the specific variables. The number of samples not analyzed ranged from 4% for respirable particle concentrations to 12% for urinary cotinine. The high number of samples not analyzed for cotinine occurred because it was measured after all other urine assays, and there was often an insufficient quantity of urine remaining. Mutagenicity was not measured with S9 in 6% of samples and without $9 in 10% of samples, in most cases because of an inadequate quantity of urine.

Diesel Exhaust and ETS Exposure

A total of 303 (99% of collection days) personal exposure samples were collected. However, 8 samples were missing particulate mass values due to filter damage and 23 were missing nicotine values. Use of medians for exposure variables (Table 2) adjusts for the skewness commonly seen in the distribution of airborne exposures, which was also present in our data.

The carmen had the highest exposures to RSP, even though they have no diesel exhaust exposure. This resulted in part because some of them were exposed to particulate matter generated by welding or by using acetylene torch for cutting and burning chassis of train cars during repairs, an activity that could occupy 1/2 to 4 hr per day. A large difference in RSP was seen in the average exposures of the carmen welders and nonwelders (Table 2), confirming the contribution of welding-associated exposures to the RSP exposures among carmen.

Despite the expected difference in diesel exhaust exposure between the shop workers and the clerks, the RSP concentrations of these job groups (Table 2) were similar, which is consistent with our previous findings from U.S. railroads . This similarity of RSP concentrations between job groups was largely due to the RSP mass contributed by ETS for the clerks. Median concentrations of ETS were highest among the carmen, who had the highest prevalence of smoking, with high concentrations also present among the clerks and the engineers.

The relative ranking of the jobs by ARP (Table 2), excluding carmen, was close to that expected by the relative contact these groups have with operating diesel locomotives, assuming that all workers are exposed to a background air concentration of about 30 p,g/m3. Specifically, highest ARPs were seen for the shop workers, who have the highest exposure to diesel exhaust, and lower ARP levels were obtained for the other job categories, which have little or no diesel exposure. This ranking and ARP levels are similar to those obtained in our previous study of railroad workers . However, this cannot explain all of the differences seen between the job groups.

Daily exposures for individual study days were combined into strata of ARP for regression analyses of urinary mutagenicity and diesel exposure. Workers in groups known a priori not to be diesel exposed (clerks, carmen) were placed in the unexposed strata . Cutoff values for the strata of ARP for smokers and non-tobacco users were 77 and 155 ug/m3, selected to create approximately equal-size strata. The median levels of ARP for the low, intermediate, and high strata of nonsmoking diesel-exposed workers analyzed by regression were 50, 110, and 181 ug/m3, respectively. A similar relationship between ARP and diesel exposure was seen for the smoking workers, with median levels of ARP in the three strata of 10, 115, and 222 ug/m3.

Urinary Mutagenicity and Diesel Exhaust Exposure

Urinary mutagenicity was first evaluated by job group among nonsmokers (Table 3). No association was present for job grouping and urinary mutagenicity +$9 according to a priori classification of diesel exhaust exposure. Highest levels of urinary mutagenicity among the nonsmokers were seen in the carmen welders, probably due to their welding exposures. Similarly, no association was present among cigarette smokers for mutagenicity +-S9 and diesel exposure as estimated by job category (Table 3).


Regression models were run separately for smokers and for nonsmokers, for mutagenicity +S9 and - S9 . In all multiple regressions, the experiment mean response was adjusted for the positive control mutagen 2-aminofluorene. Score tests for mathematical transformations of mutagenicity uncovered none superior to the untransformed value, which was used in all analyses. Cook's D influence statistic identified no more than two unduly influential observations for any analysis, and these were excluded from the final models.

Among cigarette smokers, smoking was controlled for by urinary cotinine (corrected for creatinine) and by the number of cigarettes smoked on the study day up to the time o f sample collection. These two smoking indicators have differing biologic significance: cotinine concentration is influenced by cigarette smoking prior to the study day, while the number of cigarettes smoked on the study day reflects more recent smoking exposure. Although urinary cotinine and cigarettes smoked on the day o f study were correlated (p = 0.38, p < 0.001), each contributed at p < 0.05 to the regression equation.

Independent variables for known diesel exposure status (yes = shop worker, braker, or engineer; no = clerk or carmen) and the differential effect (interactions) of diesel exposure status and respirable particles or air nicotine concentration were not significant as predictors of mutagenicity +S9, either individually or jointly (model not shown). A similar absence of diesel effect was observed in the regression for mutagenicity - S9 among cigarette smokers.

The mean mutagenicity +S9 among cigarette smokers unexposed to diesel (clerks, carmen), adjusted for urinary cotinine, ambient nicotine (ETS), and the standard mutagen was 3.18 revertants/p~mole creatinine (Table 4). Among the smoking diesel-exposed subjects, the adjusted mean mutagenicities for the three strata of ARP (low to high) were 3.12, 3.93, and 3.54, respectively ( x z = 0.48, p > 0.05). N o n e of the differences among the three strata of diesel-exposed workers or comparisons with the unexposed workers was statistically significant. Regressions were also run excluding all carmen, with results similar to those presented (results not shown).

Mutagenicity -S9 also failed to demonstrate a dose-response relationship with diesel exposure strata. The mean level of adjusted mutagenicity ( - $ 9 ) in the non-diesel-exposed and three strata of ARP (low to high) among cigarette smokers were 0.48, 0.20, 0.20, and 0.34, respectively (Table 4). As with the results with S9, there was no suggestion of an association between diesel exposure and mutagenicity without S9 among cigarette smokers.

Among non-tobacco users, multiple regressions using stratification of ARP also revealed no evidence of a dose response for mutagenicity with or without $9 (Table 4). Mean adjusted urinary mutagenicities ( + $ 9 ) , for the non-diesel-exposed and low, medium, and high strata of ARP (diesel) were 0.46, 0.37, 0.34, and 0.12, respectively (Table 4). A similar absence of dose response was seen among non-tobacco users for mutagenicity - S9 . Similarly, in the full regression models (not shown), no additional contribution was made to mutagenicity +-S9 by dichotomous diesel exposure status (yes/no) or by the interaction of diesel exposure status (yes/no) with respirable nicotine or with total respirable particles.

Among non-tobacco users the estimated between-person variances were approximately one-fourth the magnitude of the within-person (between-sample) variances, indicating a definite lack of independence in observations taken from the same worker. To examine more closely the possible effects of diesel exposure independent of individual differences and the effects of ETS, we sought to identify a subset of workers who (1) had j o b s with potentially high diesel exposure, (2) were nonsmokers, (3) were exposed to little or no ETS (based on nicotine on personal samplers), and (4) had been sampled repeatedly. Three repair shop workers met these criteria, each of whom had been sampled on 8 to l0 different days.

Plots for the repeated samples on these three workers of urinary mutagenicity +-S9 (revertants per micromole of creatinine) versus the marker of diesel exposure (ARP) showed no suggestion of a positive association (Figs. 1 and 2). This provides additional support for the regression results showing no association between diesel exposure and urinary mutagenicity among nonsmokers.

The "protective food" variable (ingestion of cabbage, brussels sprouts, or fish in the prior 2 days) was associated with lower urinary mutagenicity only with $9 among smokers and non-tobacco users, but inclusion of this variable did not alter the absence of an association of diesel exposure category and mutagenicity (data not shown).
