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Mutation Research/Genetic Toxicology and Environmental Mutagenesis
journal homepage www.elsevier.com/locate/gentox Community address www.elsevier.com/locate/mutres
A method for assessment of the genotoxicity of mainstream cigarette-smoke by use of the bacterial reverse-mutation assay and an aerosol-based exposure system
qi Joanne Kilforda, David Thorneb, Rebecca Payne3, Annette Dalrympleb, Julie Clements'1 Clive Meredithb, Debbie Dillonb'*
a Covance Laboratories Ltd., Otley Road, Harrogate, North Yorkshire HG3 1PY, UK b British American Tobacco, Group R&D, Southampton, Hampshire SO15 8TL, UK
ARTICLE INFO
ABSTRACT
20 21 22
Article history:
Received 6June 2013
Received in revised form 6 February 2014
Accepted 5 April 2014
Available online xxx
Keywords:
Tobacco whole smoke Vitrocell VC 10 Ames
Dose measurements QCM
To date there are no widely accepted methods for the toxicological testing of complex gaseous mixtures and aerosols, such as cigarette smoke, although some modifications to the standard regulatory methods have been developed and used. Historically, routine testing of cigarettes has primarily focused on the particulate fraction of cigarette smoke. However, this fraction may not accurately reflect the full toxicity and mutagenicity of the smoke aerosol as a whole, which contains semi-volatiles and short-lived products of combustion. In this study we have used a modified version of the bacterial reverse-mutation (Ames) assay for the testing of mainstream smoke generated from 3R4F reference cigarettes with a Vitrocell® VC 10 exposure system. This method has been evaluated in four strains of Salmonella typhimurium (TA98, TA100, YG1024 and YG1042) and one strain of Escherichia coli (WP2 uvrA pKM101) in the absence and presence of a metabolic activation system. Following exposure at four concentrations of diluted mainstream cigarette-smoke, concentration-related and reproducible increases in the number of rever-tants were observed in all four Salmonella strains. E. coli strain WP2 uvrA pKM101 was unresponsive at the four concentrations tested. To quantify the exposure dose and to enable biological response to be plotted as a function of deposited mass, quartz-crystal microbalances were included in situ in the smoke-exposure set-up. This methodology was further assessed by comparing the responses of strain YG1042 to mainstream cigarette-smoke on a second VC 10 Smoking Robot. In summary, the Ames assay can be successfully modified to assess the toxicological impact of mainstream cigarette-smoke.
© 2014 Published by Elsevier B.V.
24 1. Introduction
25q2 The bacterial reverse-mutation assay, also known as the Ames
26 assay [1], is widely used for the initial genotoxicity screening
27 of pharmaceuticals and chemicals. The methodology is dictated
28 by clear international regulatory guidelines (e.g. OECD, ICH) to
29 ensure consistent testing across laboratories [2,3]. Although these
30 guidelines are suitable for the testing of compounds as solutions
Abbreviations: AAN, 2-aminoanthracene; B[a]P, Benzo(a)pyrene; DMSO, dimethyl sulphoxide; FI, fold increase; ISO, international standards organisation; MR, mean revertants; NaN3, sodium azide; NQO, nitroquinoline oxide; PBS, phosphate buffered saline; PM, particulate material; QCM, quartz crystal microbalance; VC 10, Vitrocell® smoking robot; VP, vapour phase; 2NF, 2-nitrofluorene. * Corresponding author. Tel.: +44 2380 793717; fax: +44 2380 588856. E-mail address: Debbie_Dillon@bat.com (D. Dillon).
http://dx.doi.org/10.1016lj.mrgentox.2014.04.017 1383-5718/© 2014 Published by Elsevier B.V.
or suspensions, modifications are required to enable the testing 31
of pure gases [4]. The testing of complex gaseous mixtures and 32
aerosols such cigarette smoke poses even greater challenges. A 33
method capable of testing gases or aerosols would therefore be 34
very beneficial, particularly to the tobacco industry where there is 35
increased interest in performing toxicological testing on the entire 36
smoke aerosol. The development of such methods for the testing 37
of volatile tobacco products was discussed by the Committee on 38
Mutagenicity in 2009 and the absence of an adequately validated 39
methodology was noted [5]. 40
Cigarette smoke is a complex aerosol made up of both a par- 41
ticulate fraction and a vapour phase (VP), making it exceptionally 42
difficult to test this mixture by use of standard methods. To date, 43
most toxicological testing of cigarette smoke has relied heavily on 44
testing the particulate fraction with standard assays [6,7]. To cap- 45
ture the particulate matter (PM), cigarettes can be smoked onto a 46
Cambridge filter pad and the PM extracted with dimethyl sulfoxide 47
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(DMSO), which can then be treated as a standard test-article [8-10]. The results of such testing have demonstrated significant concentration-related increases in cytotoxicity and mutagenicity in several standard genotoxicity assays [11-14]. The particulate phase, however, is a small fraction of the whole-smoke aerosol [15] and testing of the particulate fraction alone does not account for semi-volatiles or gases found in the vapour phase of cigarette smoke. Some attempts have been made to test a more representative sample of whole smoke by bubbling the resulting vapour phase through phosphate-buffered saline (PBS) and testing both the particulate and vapour fractions either independently or as a mixture [8,16]. However, collecting and testing of particulate and vapour fractions independently is not ideal as it does not account for insoluble compounds or short-lived chemicals resulting from combustion. Due to the complexity of potential chemical interactions within and between phases, it is important for future toxicological testing of cigarettes to develop methods that enable testing of the entire smoke aerosol.
Recent technological advancements have seen the introduction of in vitro smoking machines, paired with exposure modules that allow exposure of cells to the whole-smoke aerosol at an air-liquid or air-agar interface. One example is the Vitrocell® VC 10 Smoking Robot which dilutes mainstream cigarette-smoke into a constant flow of air. A sample of this diluted smoke is pulled by vacuum into the exposure module where it is delivered to each chamber across the module [17]. The flow rate of the diluting air can be adjusted to alter the concentration of smoke delivered.
Dosimetry tools are also being developed that can be used in conjunction with whole-smoke exposure systems to quantify the deposition of smoke particulate mass [18]. Characterisation of quartz-crystal microbalances (QCMs) with the VC 10 Smoking Robot has demonstrated that this technology can be used to quantify particle deposition during whole-smoke exposure across a wide range of smoke dilutions [17]. The VC 10 together with QCM technology represents an appropriate system to modify the standard regulatory assays for exposure to aerosols such as whole smoke. In addition, the use of QCMs provides a valuable opportunity to monitor machine performance and present data against a quantitative dosimetry measure that could be translatable between laboratories.
In this study, we have applied a modified plating method, comparable to that reported by Araki et al. [19] and Aufderheide and Gressman [20], with minor modifications to adapt the standard regulatory Ames assay to exposure to gaseous aerosols on the Vitrocell® VC 10 exposure system. This exposure method has been evaluated with mainstream cigarette-smoke generated from 3R4F reference cigarettes on the Vitrocell® VC 10 Smoking Robot. The standard regulatory Ames assay may include a total of five bacterial strains, each of which identifies mutagens of a different chemical class [21]. The current OECD guidelines [2] recommend a total of five bacterial strains. Four strains of Salmonella typhimurium (TA1535; TA1537 or TA97 or TA97a; TA98 and TA100) are proposed, which between them detect both frameshift and base-pair substitution mutations, with GC base pairs at the primary reversion-site. A fifth strain is recommended to detect mutagens that may not be captured by the previous strains, such as certain hydrazines, oxidizing mutagens and cross-linking agents. Either Salmonella typhimurium strain TA102 or an Escherichia coli (E. coli) strain (WP2 uvrA or WP2 uvrA (pKM101)) are accepted as the fifth strain, each of which have an AT base-pair at the primary reversion site [2].
We tested the genotoxicity of whole smoke with five strains of bacteria. These included three strains accepted under the regulatory guidelines, i.e. Salmonella typhimurium TA98 and TA100, and Escherichia coli strain WP2 uvrA (pKM101). In addition, derivatives ofTA98 (YG1024) andTA100 (YG1042) were selected based on their
increased sensitivity to nitroarenes and aromatic amines [22,23], which are known mutagens present in cigarette smoke [24-26].
In the standard plate-incorporation method, bacteria are embedded in a top agar together with the test material. However, for the testing of whole smoke, it is important that the bacteria are fully exposed to both particulate and gaseous components that cannot be incorporated into the top agar and, as a result, exposure must be facilitated at the agar surface. The spread-culture method [19,20] was therefore selected for this study.
In this study we demonstrate that a modified Ames assay, similar to that used by Aufderheide and Gressman [20], with some modifications can work successfully in conjunction with the Vitrocell® VC 10, a commercially available aerosol-based exposure system. We have tested five bacterial strains with different sensitivities to mainstream cigarette-smoke. In combination with QCM technology, this method could provide an appropriate system for measurement of the toxicological impact of whole mainstream cigarette-smoke, rather than relying on testing of particulate matter and vapour phase as independent fractions.
2. Materials and methods
2.1. Chemicals and reagents
All chemicals and reagents were obtained from Sigma-Aldrich (Gillingham, UK) unless otherwise stated.
2.2. Reference cigarettes
3R4F reference cigarettes were obtained from the University of Kentucky, Kentucky, USA. Prior to smoking, cigarettes were conditioned for at least 48 h and no more than 10 days at 22 ± 1 °C and 60 ±3% relative humidity according to the International Organisation of Standardisation (ISO) 3402:2000).
2.3. Bacterial strains and culture conditions
The details of the bacterial strains used in this study are presented in Table 1.
Frozen cultures of each strain were checked for strain characteristics according to Maron and Ames and De Serres and Shelby [28,29]. As is standard procedure within Covance Laboratories, overnight cultures were prepared from frozen stocks in 30 mL nutrient broth containing appropriate antibiotics to maintain plasmids, and grown in a shaking incubator at 37 °C for 8 h.
2.4. Vitrocell® VC 10 setup and whole-smoke exposure
The Vitrocell® VC 10 Smoking Robot (Serial Number VC10/090610) was used to expose bacteria to mainstream cigarette-smoke generated from 3R4F reference cigarettes. Cigarettes were smoked according to ISO 3308:2000 (one 35-mL puff per 60 s, over 2 s), with an 8-s exhaust. Triplicate bacterial plates were exposed in Vitrocell® AMES 4 stainless steel exposure-modules with a QCM in the fourth position to record deposition of PM as a measure of dosimetry. The trumpet height in the modules was set to 2 mm above the agar or QCM surface. Mainstream cigarette-smoke was diluted into a constant stream of air with a fixed vacuum of 5 mL/min to pull the diluted smoke through the exposure module. Different concentrations of smoke were achieved by varying the flow rate of the diluting air (1.0, 4.0, 8.0 and 12.0 L/min). Modules were exposed to a total of three or eight cigarettes smoked over 24 or 64 min, respectively, with a continual flow of diluting air between each cigarette puff. At the end of the whole-smoke exposure period, the final deposited mass reading on each QCM was recorded once a plateau in the deposition curve was observed.
2.5. Ames reverse-mutation assay
The Ames assay used in this study differs from the standard method in several key aspects, for example: the agar-plate size has been scaled down from 85 to 35 mm to facilitate aerosol/whole-smoke exposure (incorporation of agar plates into the Vitrocell® Ames module, or commercially available equivalent). As a result, cofactor preparation is an exact scaled-down equivalent. Positive control concentrations per plate have also been factored and assessed for this new scaled-down plated version of the assay (Table 2). In addition, in our protocol we have included a drying step, where plates were wrapped in parafilm, vented and incubated at 37 °C for 3 days. Parafilm wrapping (with vent holes) was used to prevent agar shrinkage and dehydration. Finally, we have varied the diluting air within the exposure system to create smoke dilutions and ultimately a dose-response. This is in contrast to other studies that have used cigarette numbers to create a dose-response [20], but still achieving the same end.
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Table 1
Strain characteristics.
Bacterial strain Mutation Plasmids and characteristics Reference
S. typhimurium TA98a Frameshift (hisD3052) pKM101 bio, rfa, uvrB Ames et al. 1975 [1]
S. typhimurium TA100b Mis-sense (hisG46) pKM101 bio, rfa, uvrB Ames et al. 1975 [1]
S. typhimurium YG1024c Frameshift (hisD3052) pKM101; pYG219 bio, rfa, uvrB O-acetyltransferase-overproducing derivative ofTA98 Watanabe et al. [22]
S. typhimurium YG1042c Mis-sense (hisG46) pKM101; pYG233 bio, rfa, uvrB Nitroreductase and O-acetyltransferase-overproducing derivative ofTA100 Hagiwara et al. [23]
E. coli WP2 uvrA pKM101d Mis-sense (trpE56) pKM101 uvrA McMahon et al. [27]
Source of strains: (a) UKNCTC; (b) Covance Laboratories Inc., USA; (c) National Institute of Health Sciences, Japan; (d) Moltox Inc., USA.
Table 2
Positive control concentrations used for all strains in the presence or absence of S-9 metabolic activation.
Bacterial strain Positive control concentration (|xg/plate)
Without S-9 activation With S-9 activation
TA98 2NF 0.4 B[a]P 0.8
TA100 NaN3 1.0 AAN 0.4
YG1024 2NF 0.025 B[a]P 0.8
YG1042 NaN3 1.0 AAN 0.025
WP2 uvrA pKM101 NQO 0.16 AAN 2.5
Abbreviations: B[a]P, Benzo(a)pyrene; 2NF, 2-nitrofluorene; AAN, aminoanthracene; NaN3, sodium azide; NQO, nitroquinoline oxide.
179 Exposure of scaled down 35-mm plates (Grenier Bio-One, UK) to whole smoke
180 were performed in the absence and presence of an exogenous metabolic activa-
181 tion system, i.e. Aroclor 1254-induced rat liver S-9 from male Sprague-Dawley rats
182 (MoHox®, MolecularToxicology, Inc., USA). The plating methodology employed was
183 comparable to that published by Aufderheide and Gressman [20]. Briefly, approxi-
184 mately 2 x 107 cells were mixed with 75 |xL sodium phosphate buffer (pH 7.4) or a
185 10% S-9 mix, prepared according to Ames et al. [1] (complemented with 40 |xg/mL
186 histidine and 48.8 |xg/mL biotin for S. typhimurium, or 1.72 |xg/mL tryptophan and
187 0.41 |xg/mLcasamino acids (as detailed by Green et al. [30]) for E. coli,)to a final vol-
188 ume of 95 |xL The bacterial cell suspension was plated directly onto Vogel-Bonner
189 agar and the culture was spread over the surface of the agar. The inoculum drying
190 time for the plates was kept to a minimum (typically around 20min) and therefore
191 the effect on the metabolic activity of the S-9 mix is minimal, as confirmed by the
192 positive control data. Plates were then incubated at 37 °C until dry before transfer to
193 the Vitrocell® AMES 4 exposure modules. The actual number of plated bacteria was
194 confirmed by viability plating. Concurrent negative (air and untreated) and positive
195 controls were included with each exposure. Air controls were exposed to a constant
196 flow of synthetic air (0.2 L/min, 5 mL vacuum). Untreated and positive controls were
197 maintained at room temperature forthe duration ofthe exposure. Indeed, all control
198 data confirm the sensitivity ofthe assay and the preservation during the drying step
199 ofthe rapidly dividing and log-phase nature ofthe bacterial culture in the inoculum, 2oq3 without it being compromised in any way (Table 3).
201 Triplicate plates were used for all treatments and experiments were performed
202 six times for responding strains and three times for non-responding strains.
2.6. Data evaluation and acceptance criteria
Plates were scored with an automated colony counter (Sorcerer Image Analyser, Perceptive Instruments, Haverhill, UK) and the background lawn was inspected for signs oftoxicity, which were defined asa marked reduction in revertant numbers or a thinning ofthe background bacterial lawn. Vehicle and positive-control ranges for the standard regulatory Ames assay (on 85-mm plates) are held at Covance Laboratories Ltd. These ranges are based on a large volume of data and are consistent with accepted spontaneous revertant ranges [29,31]. These ranges were scaled down to give equivalent colony counts on 35-mm plates. An observed range of spontaneous revertant numbers for treatments performed with the scaled-down spread-plate methodology was also generated for all control treatments based on three independent experiments. These data were comparable to the scaled-down historical control data for each strain (or the parent strain where historical control data were not available, i.e. forYG1024 and YG1042). Experiments were considered acceptable if the untreated, air and positive-control mean revertant-numbers were comparable with both the observed ranges and the scaled-down historical control ranges for each strain (or the parent strain where historical control data were not available). Data were evaluated by use of both fold-increase in revertant numbers and Dunnett's test as a statistical measure. A mutagenic response was considered to be a reproducible, concentration-related increase in revertant numbers of at least 2-fold (3-fold for <10 spontaneous revertants) the concurrent air control that was significant at the 1% level (p <0.01) using Dunnett's test.
3. Results
Four strains of Salmonella typhimurium (TA98, TA100, YG1024 and YG1042) and one strain of Escherichia coli (WP2 uvrA pKM101) were exposed to diluted 3R4F mainstream smoke generated by the Vitrocell® VC 10 Smoking Robot, in the absence and presence of metabolic activation (S-9).
3.1. Culture optimisation for whole-smoke exposure
To ensure that bacteria were in an actively dividing state when plated, and that an adequate number of cells were exposed to the whole-smoke aerosol, growth-curve assessments were performed on all strains by measuring optical density and plating for viable
210 211 212
220 221 222
Table 3
Scaled-down and spontaneous revertant numbers for a 35-mm plate format.
Bacterial strain
TA98 TA100 YG1024 YG1042 WP2 uvrA pKM101
Scaled-down ranges 2.5-9.7a 12.0-28.5a 3.9-11.5a 25.5b 20.0-46.3a
Observed rangesc 3-16 9-31 1-11 9-24 22-53
a 99% reference ranges from laboratory historical data, scaled down from standard 85-mm plates to that applicable for 35-mm plates.
b Insufficient laboratory historical data to generate a range. Value presented is scaled down mean spontaneous revertant plate count stated by Hagiwara et al. [23]. c Laboratory maximum and minimum observed revertant plate counts from 35-mm spread-plate methodology treatments.
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Fig. 1. Mean number of revertants (from three replicate plates) resulting from exposure to diluted mainstream cigarette-smoke generated from 3R4F reference cigarettes in four strains of S. typhimurium and one strain of E. coli. All experiments were performed six times for responding strains and three times for non-responding strains, with 24-min exposures at four diluting airflow rates (12.0, 8.0,4.0 and 1.0L/min) and a constant vacuum of 5 mL/min. All data are plotted with concurrent air controls (0.2L/min, 5 mL vacuum).
236 colonies every hour for up to 12 h of incubation at 37 °C. These
237 assessments identified an optimal incubation time of 8 h to arrive
238 at a sufficient number of cells in the late log-phase of growth at the
239 time of plating.
240 3.2. Mutagenicity of whole mainstream cigarette-smoke
241 All five strains of bacteria were exposed to diluted 3R4F main-
242 stream cigarette-smoke with a constant exposure time of 24min
243 (three cigarettes). Different concentrations of smoke were achieved
244 with varying flow rates of diluting air (1.0, 4.0, 8.0 and 12.0 L/min).
245 Positive and negative controls, in the form of flowing synthetic
246 air (0.2 L/min, 5 mL/min vacuum) and untreated controls, were
247 included with each experiment. The data collected for positive,
248 air and untreated controls in each experiment were comparable
249 to both the scaled-down historical control ranges for the standard
250 plate-incorporation assay held at Covance Laboratories Ltd., and
251 the observed ranges generated for spread-plate methodology. In
252 addition, the data showed that there was no significant difference
253 in the range of spontaneous revertant numbers on untreated and
254 flowing air-exposed plates.
255 For all treatments in the presence of S-9, experiments were
256 performed six times for responding strains and three times for non-
257 responding strains, to assess the reproducibility of the exposure
258 method.
Following exposure to diluted 3R4F mainstream cigarette- 259
smoke in the presence of S-9, no evidence of toxicity in the form of a 260
thinning of the background bacterial lawn was observed. However, 261
with some strains (TA98 and TA100) a slight reduction in revertant 262
numbers was noted at the highest treatment concentration tested 263
(1.0 L/min diluting air). Concentration-related increases in rever- 264
tant numbers were observed in all four Salmonella strains (Fig. 1). 265
The plasmid-containing derivative of TA98 (YG1024) showed the 266
highest sensitivity to whole smoke, with a mean increase of 267
24.8-fold at the highest smoke concentration (1.0 L/min diluting 268
air) tested, when compared with the concurrent air control. The 269
increases in revertant numbers in at least the top two smoke con- 270
centrations tested (4.0 and 1.0 L/min diluting air), in all six replicate 271
experiments, were statistically significant when analysed at the 272
1% level using Dunnett's test (p < 0.01). Strong responses to whole 273
smoke were also observed in strains TA98 and YG1042 with max- 274
imum mean increases of 5.6- and 5.5-fold, respectively, compared 275
with the concurrent air control. These increases were statistically 276
significant at the two highest test concentrations (4.0 and 1.0 L/min 277
diluting air). The response of strain TA100 to diluted 3R4F main- 278
stream cigarette-smoke was lower than 2.0-fold (maximum mean 279
increase, 1.7-fold). However, statistically significant increases were 280
observed at 4.0- and/or 1.0-L/min dilutions of whole smoke in 281
four out of six replicate experiments. TA100 was therefore consid- 282
ered to produce a weak mutagenic response to 3R4F mainstream 283
cigarette-smoke following a 24-min exposure. A statistically 284
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Table 4
Average mean number of revertants (MR), from three (E. coli WP2 uvrA pKM101) or six (Salmonella strains) replicate experiments, with standard deviation (SD), and average fold-increase in revertants (FI) following 24-min exposure at four dilutions of3R4F mainstream cigarette-smoke (12.0,8.0,4.0 and 1.0L/min) in the presence ofS-9 metabolic activation. An asterix (*) indicates where statistically significant increases in revertant numbers were observed when analysed at the 1% level with Dunnett's test (p <0.01). The number in brackets indicates the number of replicate experiments where statistically significant increases were observed.
Diluting airflow (L/min) TA98 TA100 YG1024 YG1042 WP2 uvrA pKM101
MR ±SD FI MR ±SD FI MR ±SD FI MR ±SD FI MR ±SD FI
Air control 5.6 ±1.8 - 18.9 ±4.5 - 6.6 ± 1.7 - 14.8 ±2.6 - 31.3 ±4.7
12.0 9.2 ±3.2 1.9 18.6 ±2.7 1.0 23.8 ± 5.5 3.9.(2) 21.2 ±7.0 1.4 33.8 ± 3.9 1.1
8.0 15.4 ±4.3 3.1*(1) 23.5 ± 8.4 1.3*(1) 66.2 ± 15.6 10.5*(4) 30.2 ± 4.4 2.1*d) 34.5 ± 4.6 1.1
4.0 29.1 ± 6.7 5.6*(6) 30.4 ± 7.9 1.7*(3) 133.9 ± 20.0 21.7*(6) 61.1 ± 10.8 4.2.(6) 37.3 ±8.5 1.2
1.0 27.8 ±11.1 5.0*(5) 29.8 ± 4.2 1.7*(3) 152.6 ±43.6 24.8*(6) 79.2 ±5.9 5.5.(6) 32.9 ±5.5 1.1
significant increase was also observed in strain TA100 in one experiment at 8.0 L/min dilution. However, when compared against the other five replicate experiments, the response was uncharacteristically high and was, therefore, considered to be an anomalous result. In comparison to the other three Salmonella typhimurium strains, slightly higher variability between the replicate experiments was observed in TA100. No response to whole smoke was observed over a 24-min exposure period following three replicate exposures in E. coli strain WP2 uvrA pKM101 (Table 4).
As strains TA100 and E. coli WP2 uvrA pKM101 demonstrated a weak response or no response to a 24-min exposure to 3R4F whole smoke (three cigarettes), these strains were further tested for 64 min (eight cigarettes) (Fig. 2). This extended exposure period did not significantly increase the magnitude of the response observed in strain TA100 (mean increase 2.2-fold, at 4.0 L/min) although overall, there were fewer fluctuations in the results when compared with the 24-min data. Statistically significant increases were observed at 4.0 and/or 1.0 L/min dilutions of whole smoke in all sixTA100 64-min exposure experiments when analysed by use of Dunnett's test (p < 0.01). E. coli strain WP2 uvrA pKM101 remained unresponsive to whole smoke, even after the longer exposure time. These data, therefore, confirm that a 24-min exposure period is sufficient to detect a response to whole smoke in the tester strains, however, in weak-responding strains, such as TA100, more consistent data may be obtained with a slightly longer exposure time.
Exposures to diluted 3R4F mainstream cigarette-smoke were also performed in all five bacterial strains in the absence of S-9, with an exposure time of 24 min (three cigarettes).
Following duplicate experiments in each strain at flow rates of the diluting air of 1.0, 4.0, 8.0 and 12.0 L/min, no evidence of tox-icity was observed and there were no clear concentration-related increases in revertant numbers in response to diluted 3R4F mainstream cigarette-smoke (Tables 5 and 6).
3.3. Dosimetry for whole-smoke exposure
A quartz-crystal microbalance (QCM) was included in situ of all whole-smoke exposures in order to quantify the dose delivered by
measuring deposition of particulate mass. The data provided by the QCMs were used as a quality check to monitor each exposure. The final response was compared against the total deposited mass taken at the end of exposure. The data from exposures in the presence of S-9 demonstrated that in all cases where an increase in biological response was observed (in the four Salmonella strains), this correlated with an increase in particulate deposition (Fig. 3 and Table 7).
3.4. VC 10 comparison
To further test the methodology, YG1042 was exposed to 3R4F mainstream cigarette-smoke generated by a second VC 10 Smoking Robot (VC 10 (2), Serial Number: VC10/221211). Triplicate exposures were performed in the presence of S-9 only (Fig. 4). When compared, the responses on VC 10 (1) and VC 10 (2) were extremely consistent with mean increases of 5.4- and 5.5-fold, respectively, at the highest smoke concentration tested (1.0 L/min diluting air). This confirms that this Ames method can be used to generate reproducible data, irrespective of the VC 10 Smoking Robot used for smoke generation.
4. Discussion
The standard battery of genotoxicity assays used for the testing of pharmaceuticals and chemicals is governed by international regulations and guidelines (e.g., ICH and OECD) [2,3] which - with some suggested modifications - are suitable for the testing of most compounds. However, these modifications are not necessarily appropriate for the testing of complex gaseous mixtures or aerosols. Historically, the particulate and gas/vapour-phase fractions of complex aerosols have been separated and tested for cytotoxicity and mutagenicity either independently or as recom-bined fractions [32-34]. However, this may not yield an accurate representation of the genotoxicity of the aerosol as a whole, in which there may be complex interactions between chemicals in each phase, semi-volatiles or short-lived products of combustion. A more appropriate exposure method is therefore required.
Fig. 2. Mean number of revertants (from three replicate plates) resulting from exposure to diluted mainstream cigarette-smoke generated from 3R4F reference cigarettes in S. typhimurium strain TA100 and in E. coli WP2 uvrA pKM101. Experiments were performed six times for responding strains and three times for non-responding strains, with 64-min exposures at four diluting airflow rates (12.0, 8.0,4.0 and 1.0 L/min) and a constant vacuum of 5 mL/min. All data are plotted with concurrent air controls (0.2 L/min, 5 mL vacuum).
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Table 5
Average mean number of revertants (MR) from three (E. coli WP2 uvrA pKM101) orsix(TA100) replicate experiments, with standard deviation (SD), and average fold-increase in revertants (FI) following 64-min exposure at four dilutions of3R4F mainstream cigarette-smoke (12.0, 8.0,4.0 and 1.0 L/min) in the presence of S-9 metabolic activation. An asterix(*) indicates where statistically significant increases in revertant numbers were observed when analysed at the 1% level with Dunnett's test (p < 0.01). The number in brackets indicates the number of replicate experiments where statistically significant increases were observed.
Diluting airflow (L/min) TA100 WP2 uvrA pKM101
MR ±SD FI MR ±SD FI
Air control 16.1 ± 3.9 - 29.9 ±4.7
12.0 19.8 ±4.2 1.2 33.6 ±6.8 1.1
8.0 23.8 ±4.2 1.5 35.3 ±1.7 1.2
4.0 34.5 ±8.5 2.2*<4> 33.8 ±1.7 1.2
1.0 27.6 ±6.2 1.7*<3> 33.1 ±6.7 1.1
Table 6
Average mean number of revertants (MR) from two replicate experiments, with standard deviation (SD), and average fold-increase in revertants (FI) following 24-min exposure at four dilutions of 3R4F mainstream cigarette-smoke (12.0, 8.0,4.0 and 1.0 L/min) in the absence of S-9 metabolic activation.
Diluting airflow (L/min) TA98 TA100 YG1024 YG1042 WP2 uvrA pKM101
MR ±SD FI MR ±SD FI MR ±SD FI MR ±SD FI MR ±SD FI
Air control 2.9 ±1.6 - 20.9 ±0.2 - 4.0 ± 0.5 - 19.8 ±4.5 - 34.5 ±3.1
12.0 4.2 ± 0.2 1.8 24.0 ±2.4 1.2 5.5 ± 2.1 1.4 15.3 ±0.9 0.8 36.0 ±5.2 1.0
8.0 2.5 ± 0.2 1.1 21.7 ±1.9 1.0 5.2 ± 0.7 1.3 18.0 ±1.9 0.9 35.3 ±9.9 1.0
4.0 4.2 ± 2.1 2.0 22.3 ±0.9 1.1 6.2 ± 2.1 1.6 16.5 ±1.6 0.9 42.0 ±9.9 1.2
1.0 4.2 ± 0.7 1.7 26.3 ± 8.0 1.3 6.8 ± 1.2 1.7 21.0± 1.4 1.1 43.5 ±2.1 1.3
355 Cigarette smoke is one example where there is an increased
356 demand for testing the entire native smoke aerosol. To date,
357 most routine toxicological testing of cigarettes has been per-
358 formed on the particulate fraction or on soluble components of
the gas/vapour phase, captured by bubbling smoke through PBS 359
[7,10,11,16,35]. However, as the tobacco-smoke aerosol is made 360
up of more than 6000 chemicals divided over two distinct phases 361
[36], it is -important to test the aerosol as a whole to capture 362
Fig. 3. Average mean number ofrevertants from three (E. coli WP2 uvrA pKM101) or six (Salmonella strains) replicate experiments, with standard deviation, in four strains of S. typhimurium and one strain of E. coli plotted against mean deposited mass following 24-min exposures to 3R4F mainstream smoke at diluting airflow rates of 12.0, 8.0, 4.0 and 1.0 L/min (5 mL/min vacuum).
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Table 7
Average mean number of revertants from three (E. coli WP2 uvrA pKM101) or six (Salmonella strains) replicate experiments, and associated deposited PM with standard deviation(SD), following 24-min exposure at fourdilutions of3R4F mainstream cigarette-smoke (12.0, 8.0,4.0 and 1.0L/min).
Bacterial Airflow Mean Deposition
strain (L/min) revertants (^g/cm2) + SD
TA98 12 9.2 0.01 i 0.01
8 15.4 0.10 i 0.02
4 29.1 0.57 i 0.04
1 27.8 3.16 i 0.31
TA100 12 18.6 0.02 i 0.01
8 23.5 0.09 i 0.05
4 30.4 0.57 i 0.04
1 29.8 3.00 i 0.22
YG1024 12 23.8 0.02 i 0.02
8 66.2 0.15 i 0.04
4 133.9 0.55 i 0.07
1 152.6 3.20 i 0.27
YG1042 12 21.2 0.03 i 0.02
8 30.2 0.10 i 0.03
4 61.1 0.52 i 0.07
1 79.2 2.55 i 0.60
WP2 uvrA 12 33.8 0.05 i 0.01
pKM101
8 34.5 0.07 i 0.02
4 37.3 0.48 i 0.01
1 32.9 2.69 i 0.12
the full complexity of interactions, including the volatile or insoluble components that may not be captured by testing the fractions individually. Several modifications to the standard genotoxicity methodologies have been used previously for the testing of gaseous or volatile compounds, such as exposing cells in sealed bags or culture vessels [34-42], but so far none have been widely accepted as best practice for the testing of cigarette smoke.
In this study we have used the spread-plate methodology -generally comparable with that described by Araki et al. and Aufderheide and Gressman [19,20,43] - to modify the regulatory Ames assay for the testing of mainstream cigarette-smoke with the Vitrocell® VC 10 Smoking Robot and Vitrocell® Ames exposure modules. The VC 10 dilutes mainstream cigarette-smoke into a constant flow of air, which can be adjusted to deliver different concentrations of smoke to the cells in the exposure modules. We incorporated quartz-crystal microbalances into the fourth position
Fig. 4. Comparison of average mean number of revertants resulting from exposure of S. typhimurium strain YG1042 to diluted 3R4F mainstream cigarette-smoke on two VC 10 Smoking Machines. The average mean number of revertants from three replicate 24-min exposures performed on VC 10 (2), serial number: VC10/221211, at four diluting airflow rates (12.0, 8.0, 4.0 and 1.0 L/min) and a constant vacuum of 5 mL/min, were compared with the average mean number of revertants from six replicate 24-min exposures performed on VC 10(1), serial number: VC10/090610.
of the exposure modules in order to quantify the dose by measuring particulate deposition. The Ames method was scaled-down from that described in OECD guidelines [2] by use of a spread-plate method to ensure that bacteria were directly exposed to the entire smoke aerosol at the air-agar interface. In applying this method to test aerosols, it is even more important to use actively dividing bacteria as the exposure time is much shorter in comparison with that in standard Ames methods. In the Ames plate-incorporation assay, the test article remains embedded in the agar with the bacteria for up to 72 h and, therefore, exposure is only limited by the stability of the test article and/or the metabolic activity of the S-9 fraction. In the spread-plate method of exposure to a flowing stream of diluted whole smoke, where volatiles or components of the VP in particular may not absorb into the agar, the exposure time is significantly less. We therefore assessed the growth curve for each strain to obtain a sufficient number of bacteria that were in an actively dividing state (late log-phase) at the time of plating. The exposure time and vacuum rate, which dictates the flow rate of diluted smoke passing over the cells, were kept constant for all treatments and the dose of cigarette smoke delivered to the cells was controlled by varying the flow rate of the diluting air. In this way, all parameters remained consistent except the smoke concentration itself. The applicability of this scaled-down methodology to test aerosols was confirmed by demonstrating that the spontaneous revertant rate of untreated controls was consistent with scaled-down historical control ranges for the standard plate-incorporation method held at Covance Laboratories Ltd. In addition, these revertant numbers remained relatively unchanged in bacteria that were exposed to flowing synthetic air in the exposure system.
Following 24-min (three cigarettes) exposures of five strains of bacteria to diluted 3R4F mainstream smoke in the presence of S-9, concentration-related increases in revertant numbers were observed in the four exposed strains of S. typhimurium (TA98, TA100, YG1024 and YG1042). The most responsive of these was YG1024, with a maximum mean increase of 24.8-fold when compared with the concurrent air control. YG1024 is a derivate of TA98, which carries an additional plasmid (pYG219) encoding an over-expressed O-acetyltransferase gene. It is reported to have increased sensitivity to nitroarenes and aromatic amines, which are known to contribute to the mutagenic activity of cigarette smoke [22,24-26,44]. The high sensitivity of YG1024 has been previously utilised in mutagenicity studies to compare the urine of smokers with that of non-smokers, or between smokers of different brands of test cigarettes [45,46]. In the comparison study between smokers and non-smokers, YG1024 demonstrated 7-fold higher numbers of induced revertants when compared with the parent strain TA98. When similar calculations were performed on the current data set, the response we observed for YG1024 was 6.6-fold higher than that of the parent strain.
Strong responses to whole smoke were also observed in strain TA98 (maximum mean increase, 5.6-fold) and the TA100 plasmid derivative, YG1042 (maximum mean increase, 5.5-fold), which encodes over-expressed O-acetyltransferase and nitro-reductase genes [23]. The results obtained in this study were in fact comparable with data published by Aufderheide and Gressmann in 2008 [20], who used a similar reduced-scale spread-plate methodology. The exposure dose in that study was determined by varying the number of cigarettes rather than diluting the air-flow, as we have demonstrated here. However, in the present study we observed much higher responses in YG1024 than in the parent strain TA98 (approximately 5-fold higher). We also observed 2-3-fold higher responses to whole smoke in strain YG1042 than in its parent strain TA100 (maximum increase of 1.7-fold with three cigarettes). Data from the CULTEX exposure system showed the response of YG1042 to be slightly lower than that of TA100. The stronger responses observed in strains YG1024 and YG1042 are in keeping with the
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enhanced sensitivity of these strains to mutagenic nitroarenes and aromatic amines [23,44].
In agreement with Aufderheide et al. [20] we observed that the response to whole smoke was weaker in strain TA100 than in strain TA98 and its derivative YG1024. The magnitude of the response in TA100 remained constant, even with an extended exposure time of 64min. The weaker response of TA100 is also consistent with trends observed in the testing of cigarette-smoke particulate matter, where greater increases are historically found after treatments of TA98 when compared withTA100 [8,16,47,48].
In accordance with regulatory guidelines, the E. coli strain WP2 uvrA pKM101 was also included in this study to detect mutation at an AT base-pair reversion site. Following 3R4F whole-smoke exposure in this strain over both a 24-min (three cigarettes) and a 64-min (eight cigarettes) period, no increases in revertant numbers were observed. This is consistent with the whole-smoke response of TA102, which is also an AT base-pair reversion-site strain, in the CULTEX exposure system [20]. However, with the CULTEX system Aufderheide et al. did report a small response to 2R4F mainstream cigarette-smoke in E. coli strain WP2 uvrA pKM101. Although no response was observed in our exposure system, it is considered important to include E. coli strain WP2 uvrA pKM101, or one of the alternative strains, in further investigations and cigarette-comparison studies to enable detection of additional mutagens such as cross-linking agents and hydrazines, and to maintain a strain selection with the ability to detect a wide range of mutational events.
Also in keeping with regulatory guidelines, each of the five bacterial strains was also exposed to diluted 3R4F mainstream cigarette-smoke in the absence of S-9. Following 24-min (three cigarettes) exposures, no clear concentration-related increases in revertant numbers were observed. This suggests that the products of metabolism are the main factors that cause the mutagenic activity observed in cigarette smoke. This is consistent with the test results of both TPM and whole cigarette-smoke with different exposure systems [11,16,37,43].
All experiments in this study were performed with a QCM in situ of biological exposure. The QCM is a newly emerging dosimetry tool for use with whole-smoke exposure systems which measures deposited particulate mass [17,18]. The QCM has enabled quantification of smoke exposure dose, which is important for any standard regulatory methodology and should be considered when developing a modified method for whole-smoke exposure. The deposited mass data collected in this study were used to plot the biological response to whole smoke and demonstrated that the increases in revertant numbers observed in the presence of S-9 in the four Salmonella strains correlated with increasing particulate deposition. To further develop this tool and fully incorporate it into the methodology, additional work is required to accumulate a historical range to identify the accepted variability in deposition between exposures that can then be used to dictate acceptance criteria for any intended dose. In addition, the QCM tool only measures received particulate dose, which is the minority fraction within the whole-smoke aerosol. For full dosimetry analysis, a measure of the VP fraction is also required.
In summary, these data have demonstrated good interexperiment reproducibility of the smoke-exposure methodology presented. Dose-dependent, mutagenic activity of 3R4F mainstream cigarette-smoke has been demonstrated and the exposure was quantified by measuring total deposited particulate mass. The suitability of this modified Ames method has been supported by extremely consistent data obtained with a second VC 10 Smoking Robot in strain YG1042. Machine-to-machine variability has yet to be compared for the VC 10 but these data suggest that the exposure method is appropriate and likely to be reproducible in inter-laboratory testing. The responses of each strain tested on the
VC10 are also largely comparable to those observed in studies with 511
the CULTEX exposure system with 2R4F reference cigarettes [20]. 512
This adds further confidence and evidence in support of the con- 513
cept of testing a whole-smoke aerosol by use of a scaled-down plate 514
methodology. 515
Further work, including comparisons between cigarettes with 516
different ISO tar yields could be useful in determining the discrimi- 517
nating power of the different bacterial strains. To complement this 518
work, additional Ames strains could be investigated and incorpo- 519
rated into a proposed test battery for cigarette smoke. The final 520
battery of strains used to assess cigarette smoke should capture the 521
full complement of base-pair substitution and frameshift metations 522
at both GC and AT reversion sites. In addition, some combination 523
of plasmid-enhanced YG strains should be included for the reli- 524
able detection of mutagenic activity from nitroarenes and aromatic 525
amines. 526
In conclusion, we have demonstrated that the Ames assay, 527
which is routinely used for the initial genotoxicity screening of 528
pharmaceuticals and chemicals, can be modified for testing main- 529
stream cigarette-smoke with the Vitrocell® VC 10 Smoking Robot. 530
Indeed, the methodology presented here has other applications 531
such as the testing of other gases or aerosols. 532
Conflict of interests 533
The authors report no conflict of interests. 534
Role of funding source 535
All the authors are the employees of Covance Laboratories Ltd. q4 536
or British American Tobacco, Covance Laboratories Ltd., Harrogate, 537
UK, conducted all experimental work and were funded by British 538
American Tobacco. 539
Author contributions 540
Joanne Kilford and David Thorne designed the study. Joanne Kilford and Rebecca Payne managed and conducted all experimental work. Joanne Kilford analysed the data and wrote the manuscript. Annette Dalrymple, Julie Clements, Clive Meredith and Debbie Dillon provided scientific support. All authors approved the final manuscript.
Acknowledgements
The authors would like to acknowledge Mark Ballantyne for his scientific advice and Laura Jeffrey, Adam Seymour, Jamie Young, Sally Forrest and James Wilkinson for their technical assistance.
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