Mutagenesis and Acute Toxicity Studies on Saliva ... - ACS Publications

Chapter 8

Downloaded by NORTH CAROLINA STATE UNIV on October 4, 2012 | http://pubs.acs.org Publication Date: May 5, 1997 | doi: 10.1021/bk-1997-0654.ch008

Mutagenesis and Acute Toxicity Studies on Saliva-Leached Components of Chewing Tobacco and Simulated Urine Using Bioluminescent Bacteria Shane S. Que Hee Department of Environmental Health Sciences and Center for Occupational and Environmental Health, School of Public Health, University of California—Los Angeles, 10833 Le Conte Avenue, Los Angeles, CA 90095-1772 The task of minimizing expensive chemical analyses is usually attempted through bioassay techniques or tracers to signal the presence of compounds of interest before complete chemical analysis. The Salmonella typhimurium reverse mutation assay has been the chief bioassay used. However only point mutations are determined, and metals and organics genotoxic through other mechanisms are not detected. The use of bioluminescent bacteria in acute (Photobacterium phosphoreum) and genotoxic (Vibrio fischeri dark mutant) assays has been investigated for compounds in chewing tobacco that are available to simulated saliva and whose metabolites may be present in urine (investigated in simulated urines). Gas chromatography/mass spectrometry revealed that nicotine was the major compound leached into simulated saliva. Nicotine was not genotoxic in the reverse mutation test using a dark mutant, unlike its metabolite, cotinine. Recommendations on the conditions that need to be controlled are provided for these tests. Chemical analyses of samples containing unknown compounds is expensive. Often toxicity information is the desired endpoint although monitoring for regulated pollutants is often assumed to suffice. There are millions of nonregulated pollutants whose toxicity may be potent, and, if not so, may be present at high enough concentrations to pose threats to biota. This is so for many wastewaters, landfill leachates, and polluted environments. Biological media are even more complex mixtures of endogenous compounds, xenobiotics, and their metabolites and adducts. Tracers have been used to show that pollutant discharges contribute to environ mental quality. Tracers also inform on the degree of contribution of a specific point source to a sample. However, no information is provided on toxicity. One approach is to define any medium having the tracer to be toxic no matter the dilution as the U.S. © 1997 American Chemical Society

In Environmental Biomonitoring; Subramanian, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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EPA does for a diluted listed hazardous waste. For animal bioassays, radiolabels have enabled investigation of toxicokinetics and toxicodynamics of xenobiotics without identifying the labeled species, thus avoiding tedious chemical analyses where endogenous chemicals may interfere. Attempts to identify an unknown compound lead to having to cope with the background of endogenous chemicals. The best approach is to determine toxicity directly. There are many kinds of toxicities to humans: acute (effects for up to one week after exposure), subacute (effects for up to a month or so), subchronic (effects beyond one month to a year), and chronic (effects beyond a year). In addition ecotoxicological effects to biota have different time frames and susceptibilities. In many cases non-human biota are more at risk to pollution than are humans. The most logical approach is to use chemical tests and microbial assays before plant, invertebrate, and vertebrate bioassays. The Toxi city Characteristic Leaching Procedure (TCLP) measures human bioaccessability of specific organics and inorganics of a solid waste that may potentially pollute ground and surface waters used for human drinking water. Bioassays like the Salmonella typhimurium reverse mutation assay (the "Ames Test"} are the most familiar of the microbial assays. The Ames test detects organics that induce point mutations but not metal or organic genotoxins that operate by other mechanisms(7). A battery screen ing approach is ultimately necessary if human health effects are to be predicted. Such an approach is also essential for ecotoxicological screening. There is still need for a general genotoxicity test that not only detects point mutations, but also such DNA effects as large and small deletions, inhibition of synthesis, crosslinking, and intercalation. The reverse mutation assay of a dark mutant of the bioluminescent bacterium Vibrio fischeri to regain the bioluminescent state detects all these mechanisms sensitively (2). The same instrumentation is used to evaluate acute and chronic toxicity using bioluminescent Photobacterium phosphoreum (3,4). The effective concentration at which the luminescence is 50% that of the control (EC ) is the diagnostic parameter for the acute test. The 5 to 30 min EC for the acute test (the Microtox test) is correlated to Draize Test results for eye irritation, the acute L D for Salmo gairdneri, Daphnia, Spirillum, and the 14-day LD for guppies (Poecilia reticulata) (4). The chronic test 24-h Lowest Observed Effective Concentration (LOEC) is correlated to 7-day Ceriodaphnia dubia data (3). The aims of the present paper are to review the author's research with bioluminescent bacteria in assessing the acute toxicity of simulated biological media like urine and saliva in 5.0-mL volumes using Photobacteriumphosphoreum (4-8) and genotoxicity with a dark mutant of Vibrio fischeri in O.200 mL volumes (2). The measurements were performed on the Microtox Model 500 Analyser (Microbics Corp., Carlsbad, CA) at 15°C for the acute test, and at 27°C for the genotoxicity test. 50

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Acute Toxicity Experiments Test Media. The acute test dilution medium was 2.0% saline. Extensive testing (4) showed that the optimum bioluminescence at 5, 15 and 25 min occurred over the sodium chloride range of 1.9-3.3% (ionic strengths of 0.32-0.58 M) with EC values of 1.3% and 5.0%. Thus sample sizes in toxicity testing should be kept small relative to growth medium. A saline content of 2% discourages growth of many bacterial 50


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contaminants, and allows nonsterile conditions to be used during toxicity testing. All light was quenched at pH values below 3.20, with the EC being pH 5.34. The pH optimum was between 6.5-7.5. These results showed that pH control was essential and that acid media were toxic. To demonstrate whether media truly contained toxic compounds instead of being toxic through pH or osmotic effects, the pH was kept constant in the 6.5-7.5 range and the saline concentration between 2.0-3.0% for environmental and biological media acute toxicity evaluations. The biological test media examined for acute toxicity were four standard freezedried urines and one simulated saliva. Two urines werefromthe National Institute for Standards and Technology (Gaithersburg, MD) SRM 2670 [normal (I) and spiked (II) concentrations of toxic metals]; two others were from Instrumentation Labora tory (Orangeburg, NY) with Level 1 being unspiked (I) and Level II containing patented amounts of organics and metals. Each liter of simulated saliva contained: 555 mg sodium as sodium chloride; 500 mg potassium as potassium chloride; 100 mg calcium as calcium chloride; 150 mg phosphorus as sodium dihydrogen phosphate; 25 mg magnesium as magnesium chloride; 2700 mg of mucin Type III; 88 mg urea; 200 mg D-glucose; 100 units of amylase; 700 units of lysozyme; and 4 units of phosphatase. The medium was adjusted to pH 7.0 with hydrochloric acid and sodium hydroxide. The additional substrates tested were nicotine (4), cotinine (4), straight chain aldehydes and carboxylic acids up to C (5), glyoxal and glyoxylic acid, pyruvaldehyde and pyruvic acid, and various ketones, especially methyl ketones that were straight chain up to C (8).


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Aldehydes, Ketones, and Carboxylic Acids. Quantities in the ng/mL-mg/L range of aldehydes, ketones, and carboxylic acids have been detected as ozonolysis bypro ducts of drinking water (9). Since the luciferin responsible for light emission in Photobacterium phosphoreum is w-tetradecanal, competitive inhibition of luciferase by aldehydes and carboxylic acids was expected. To make the insoluble compounds bioavailable, aldehydes beyond C , carboxylic acids beyond C , and ketones beyond C were solubilized in 0.45% methanolic growth medium. Table I gives the 25 min EC values for each class of compound (5). From C to C , the toxicity for compounds of the same number of carbon atoms decreased in the order carboxylic acid, aldehyde, and ketone. Similarly beyond C , the order was aldehyde, ketone and then carboxylic acid. The toxicities for formic, acetic, and pyruvic acids were about the same. However formaldehyde was far more toxic than acetaldehyde or butyraldehyde. In contrast acetone was far less toxic than methyl ethyl ketone. Pyruvaldehyde was far more toxic than glyoxal but not as toxic as formaldehyde. Both the C acid and aldehyde caused stimulation of bioluminescence proving that both can act as luciferins. A cocktail of aldehydes and carboxylic acids at their highest concentrations found in drinking water disinfected with ozone (9) was not toxic, indicating that the Microtox test was not sufficiently sensitive. The data show that methyl ketones also inhibit luciferase competitively just as aldehydes and carboxylic acids do. The carbonyl group in the latter is not situated too differently from that in carboxylic acids. Another indication of the importance of the position of the carbonyl group is evidenced from the fact that 3-pentanone is about three times less toxic than 2-pentanone. 3

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Table I. EC (25-Min) Values (in mM) for Some Aldehydes (ALD), Ketones (KET), and Carboxylic Acids (CAR) of Different Chain Lengths (C ) in the Acute Toxicity Test (5,8) 50

n

ALD

c


n

1 2 3 4 5 7 8 10 11 12 13 14 2-Di 3-Pyr

KET

CAR

0.227 (0.014) 6.88 (0.51) Not done 1.374 (0.011) Not done 0.101 (0.0083) Not done 0.0186 (0.0042) 0.0297 (0.0037) Not done 0.00486 (0.00051) Stimulation 7.40 (0.44) 0.540 (0.017)

0.172 (0.0076) 0.160 (0.0096) Not done 0.196 (0.0070) Not done 0.133 (0.0028) Not done 0.0523 (0.0052) Not done 0.0163 (0.0011) 0.0216(0.0062) Stimulation 0.127 (0.0029) 0.151 (0.0039)

Not applicable Not applicable 243 (19) 51.9(2.7) 5.30 (0.36) 0.360 (0.047) 0.0781 (0.011) 0.0275 (0.0026) Not done Not done 0.0070 (0.0014) Not done Not applicable Not applicable

a

Methyl ketones with the carbonyl group at the 2-position. ALD beyond C , CAR beyond C , and KET beyond C are dissolved in 0.45% methanolic saline growth medium. Di, two carbonyls or carboxylic acid groups. Pyr, pyruvaldehyde or pyruvic acid. The values in parentheses are standard deviations of the triplicate measurements. 3

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Urine Experiments. The two SRM urines on reconstitution had pH values of 4.54.8, and were toxic because of the acidity. These urines were henceforth adjusted to pH 6.5. This caused a twenty-fold toxicity decrease. However the supposedly more toxic Level II SRM was less toxic (25-min EC of 11.6 +/-0.94 g/L) than the unspiked urine (25-min EC of 9.44 +/-0.52 g/L), correcting for creatinine. Inductively coupled plasma-atomic emission spectroscopy ICP-AES (10) showed that zinc was present in higher concentrations in the unspiked SRM (1.316 mg/L) than in the spiked (0.494 mg/L) sample, offsetting the higher concentrations of copper, lead and selenium in the spiked SRM. The zinc is a contaminant from the vial stopper. Copper (365 ng/mL) accounted for the toxicity of the spiked urine. The other two reference urines had pH values of 5.8-6.5 and therefore did not require pH adjustment. Upon correction for creatinine, the unspiked urine had a 25 min-EC value of 10.2+/-0.98 g/L whereas the spiked urine had a value of 2.57+/0.16 g/L, or four times the toxicity. ICP-AES confirmed that heavy metals were generally higher in the spiked urine than in the unspiked, especially cadmium (30 ng/mL), copper (421 ng/mL), lead (447 ng/mL), selenium (824 ng/mL), and zinc (2.7 mg/L). Ionic strengths of all urines fell within the optimum range so that any effects could not be caused by this parameter. 50

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Nicotine (25-min EC of 7.39+/-1.46 χ 10" M) was more acutely toxic than cotinine (25-min EC of 11.8+/-1.3 5 χ 10" M) in 2% saline solution. When nicotine and cotinine were spiked into the urines to produce concentrations found in the urines of tobacco users, the urine matrix was the major toxic contribution rather than the added compound so that toxicities did not differfromthose of unspiked urines. Thus unless urines are dilute enough to allow the toxicity of nicotine and cotinine to be manifested, the acute test is more sensitive to the urine matrix than to the nicotine or cotinine. Thus it is unlikely that the acute test will be a sensitive nonspecific screening test for nicotine and cotinine in urine. 50

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Saliva Testing of Chewing Tobacco Leachates. Smokeless tobacco is the chief tobacco-based alternative to smoking. Different 3 oz brands of ground chewing tobacco of weight 2 g (Redman long cut; Beechnut wintergreen; and Levi Garett) were extracted with artificial saliva at 37°C for 1 h in a shaking water bath to simulate mastication. After centrifugation and filtration through 5.0-, 2.0-, and 0.45 -pm fil ters, the pH was adjusted to 7.0 (NaOH), and the toxicity evaluated correcting for color (7). The 25-min EC values were in mg/mL: Redman, 5.30+/-0.54; Beechnut, 6.46+/-0.91; and Levi Garett, 4.80+/- 0.52, based on dissolved solid dry weights. An average of 37+/-2% of the original mass was extracted. An EPA type extraction scheme firstly at pH 7.0 (hexane; 3 extractions), then at pH 2.0 (diethyl ether; 3 extractions), and then at pH 10.0 (hexane; 3 extractions) was then perform ed. The major toxic compounds were extracted at pH 7.0 (4-5 fold increase in EC ). Gas chromatography/mass spectroscopy (GC/MS) of the pH 7.0 hexane extract revealed that the major component was nicotine, then dihydroactiniolide, lH-indole3-acetonitrile, and an unidentified trace peak. A quantitative nicotine extraction study showed it was >90% extracted with 5 extractions at pH 10.0, but at pH 7.0 even 10 extractions were only 60% efficient in spite of an asymptote for cumulative mass extracted. The quantitative extraction of nicotine at pH 10.0 removed the major part of the toxicity. ICP-AES analysis revealed that the residual toxicity in the aqueous layer was probably caused by iron (2.77 mg/L), the 15-min EC of ferric chloride being 0.8 mg/L. Thus the analysis of nicotine in saliva is feasible since the saliva medium is not toxic and the effect of nicotine dominates unlike for urine. A further study investigated whether carbonyl compounds contributed to the residual toxicity after hexane extraction at pH 10.0. This was accomplished by reaction with 0-(2,3,4,5,6)-pentafluorophenylhydroxylamine hydrochloride at pH 2.0 before hexane extraction of oximes (77). Carbonyl compounds did not cause the residual toxicity nor significant toxicity in the original leachates (6). 50

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Genotoxicity of Nicotine and Cotinine The genotoxicity experiments were carried out over 40 hours in a very complex medium (2) also containing 0.47% dimethyl sulfoxide with and without rat S9 fraction (2). Positive controls were: 2-aminoanthracene (2AA) as a S9-point mutagen; N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as a direct acting point mutagen; and phenol as a direct acting DNA intercalator which also served as the positive control for the acute test. Nicotine and cotinine and their mixtures, with nicotine added to the positive controls were evaluated. Twofold dilutions of analytes In Environmental Biomonitoring; Subramanian, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.


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were examined over the nicotine concentration range 0.07-1.6 mg/mL observed for the saliva of tobacco chewers. The same concentration range for cotinine was examined also. Genotoxicity is signalled by the reappearance of bioluminescence from a dark mutant of Vibrio fischeri Strain NRRL-B-11177 at 27°C after incubation for a specified time and at a given concentration. Nicotine was not genotoxic in the absence or presence of S9. However cotinine was a direct acting genotoxin at 1.24 mg/mL at 13-15 h incubation, and at 2.50 mg/mL between 18-30 h. Addition of S9 caused genotoxicity only at 1.25 mg/mL after 9 h. The effects of nicotine on positive controls were complex: potentiation occurred with 2AA with and without S9, phenol without S9, and cotinine with and without S9 by shifting the incubation times and concentrations for bioluminescence; and nicotine antagonized MNNG. Nicotine acts as a potentiator and antagoniser. Since nicotine (half-time, 2-3 h) is quickly metabolized to cotinine and other compounds, cotinine adverse genotoxicity may be of great importance relative to genotoxic tobacco human chronic effects. Fortunately exposure to tobacco is usually expressed through cotinine marker in biological fluids. The correlations with cotinine however assume that it is less biologically active than nicotine. At least relative to genotoxicity this is not so, and lower birth weights are correlated with increasing cotinine in body fluids (2). The effect may be direct. The role of the tobacco-specific nitrosamines (TSNA), the major known genotoxins in smokeless tobacco, is unknown with regard to their interactions with nicotine and cotinine. Conclusions The acute toxicity bioluminescence (Microtox) test cannot be used to screen nicotine and cotinine in urine, but can be used for saliva. Growth media containing environmental and biological samples must be adjusted to pH 6.5-7.5 for toxicity testing. Cotinine is a direct acting genotoxin in the Vibrio fischeri dark mutant (Mutatox) test but nicotine is not active with or without S9. Nicotine has a complex interactive effect on potent genotoxins. Literature Cited 1. Gee, P.; Maron, D.M.; Ames, B.N. Proc. Natl. Acad. Sci. U.S.A. 1994,

91, 11606-11610. 2. Yim, S.H.; Que Hee, S.S. Mutat. Research 1996, 336, 275-283.

3. Mazidji, C.N.; Koopman, B.; Bitton,G.;Voiland,G.;Logue, C. Tox. Assessment 1990, 5, 265-277. 4. Chou, C.C.; Que Hee, S.S. J. Biolumin. Chemilumin. 1993, 8, 39-48. 5. 6. 7. 8.

Chou, C.C.; Que Hee, S.S. Ecotoxicol. Environ. Safety 1992, 23, 355-363. Chou, C.C.; Que Hee, S.S. J. Agric. Food Chem. 1994, 42, 2225-2230. Chou, C.C.; Que Hee, S.S. Environ. Toxicol. Chem. 1994, 13, 1177-1186. Chen, H.F.; Que Hee, S.S. Ecotoxicol. Environ. Safety 1995, 30, 120-123.

9. Yamada, H.; Somiya,I. Ozone Sci. Eng. 1989, 11, 125-141. 10. Que Hee, S.S.; Boyle, J.R. Anal. Chem. 1988, 60, 1033-1042. 11. Cancilla, D.A.; Chou, C.C.; Barthel, R.; Que Hee, S.S. J. Assoc. Offic. Anal. Chem. Internat. 1992, 75, 842-854.


Mutagenesis and Acute Toxicity Studies on Saliva ... - ACS Publications

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