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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
The mutagenic assessment of mainstream cigarette smoke using the Ames assay: A multi-strain approach
David Thornea'*, Joanne Kilfordb, Michael Hollingsb, Annette Dalrymplea, Mark Ballantyneb, Clive Mereditha, Deborah Dillona
a British American Tobacco, Group R&&D, Southampton, Hampshire SO15 8TL, UK b Covance Laboratories Ltd., Otley Road, Harrogate, North Yorkshire HG3 1PY, UK
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ARTICLE INFO
Article history:
Received 12 November 2014 Received in revised form 1 February 2015 Accepted 3 March 2015 Available online 5 March 2015
Keywords: Whole smoke Ames VC 10 Dosimetry QCM
ABSTRACT
Salmonella typhimurium strains TA1535, TA1537, TA97, TA102 and TA104 were assessed for their suitability and use in conjunction with a Vitrocell® VC 10 Smoking Robot and 3R4F reference mainstream cigarette smoke. Little information exists on TA97, TA104, TA1535, TA1537 and TA102 using an aerosol 35 mm spread-plate format. In this study, TA1535 and TA1537 were considered sub-optimal for use with a scaled-down format, due to low spontaneous revertant numbers (0-5 revertants/plate). In the context of a regulatory environment, TA97 is deemed an acceptable alternative for TA1537 and was therefore selected for whole smoke exposure in this study. However, there is no acceptable alternative for TA1535, therefore this strain was included for whole smoke exposure. TA1535, TA97, TA102 and TA104 were assessed for mutagenic responses following exposure to cigarette smoke at varying concentrations (using diluting airflow rates of 1.0,4.0,8.0 and 12.0L/min), and exposure times of 24 and 64min. A positive mutagenic response to cigarette smoke was observed in strain TA104 at both the 24 and 64min time points, in the presence of S-9, at the highest smoke concentration tested (1.0L/min diluting airflow). The three remaining strains were found to be unresponsive to cigarette smoke at all concentrations tested, in the presence and absence of metabolic activation. Cigarette smoke particulate deposition was quantified in situ of exposure using quartz crystal microbalance technology, enabling data to be presented against an associated gravimetric mass (^g/cm2). Finally, data obtained in this study were combined with previously published Ames data for TA98, TA100, YG1024, YG1042 and Escherichia coli (WP2 uvrA pKM101), generated using the same 35 mm methodology. The combined data-set was used to propose an aerosol testing strategy, based on strain compatibility with the whole smoke aerosol, whilst maintaining the essence of the regulatory guidelines for the standard Ames assay.
© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
Abbreviations: 2NF, 2-nitroflourene; AAI, air agar interface; AAN, 2-aminoanthracene; FI, fold increase; ICH, International Commission on Harmonization; ICR-191, 6- chloro- 9- [3- (2- chloroethylamino) propylamino] -2-methoxyacridine dihydrochloride; MMC, mitomycin C; MR, mean revertants; NaN3, sodium azide; OECD, Organisation for Economic Co-operation and Development; QC, quality control; QCM, quartz crystal microbalance; SD, standard deviation; TPM, total particulate material; VC 10, Vitrocell® Smoking Robot; WS, whole smoke.
* Corresponding author. Tel.: +44 2380 58 88088; fax: +44 2380 58 88856.
E-mail addresses: David_Thorne@bat.com (D. Thorne), Joanne.Kilford@covance.com (J. Kilford), Michael.Hollings@covance.com (M. Hollings), Annette.Dalrymple@bat.com (A. Dalrymple), Mark.Ballantyne@covance.com (M. Ballantyne), Clive_Meredith@bat.com (C. Meredith), Debbie_Dillon@bat.com (D. Dillon).
1. Introduction
The bacterial reverse mutation, or Ames assay, is one of the most widely used assays for assessing the mutagenicity of chemicals in vitro. The bacterial strains used in this assay have mutations in various genes within the histidine, or tryptophan (in the case of the Escherichia coli) operon, which code for enzymes required for the biosynthesis of the amino acid histidine or tryptophan. A large number of tester strains are available with either frameshift mutations or base-pair substitutions, making the assay suitable for detecting a broad spectrum of chemicals capable of producing genetic damage and, ultimately, gene mutations [1-3]. The Ames assay has been used to determine the mutagenicity of complex chemical mixtures [4] and as such has been used to assess the mutagenicity of cigarette smoke, which has been demonstrated to have clear mutagenic activity [5]. The assay is relatively
http://dx.doi.org/10.1016/j.mrgentox.2015.03.006
1383-5718/© 2015 The Authors. Published by Elsevier B.V. This is an open access article underthe CC BY license (http://creativecommons.org/licenses/by/4.0/).
Table 1
Strain characteristics and responsiveness to cigarette smoke.
Bacterial strain Mutation Plasmid and characteristics Cigarette smoke particulate phasea Mainstream cigarette smoke aerosolb
S. typhimurium TA98 Frameshift (hisD3052) [2] pKM101 bio, rfa, uvrB [2] Positive [18-21] Positive [22-25]
S. typhimurium TA100 Mis-sense (hisG46) [2] pKM101 bio, rfa, uvrB [2] Positive Positive [23-25]
[18,20,21,26,27] Negative [22]
S. typhimurium TA1535c Mis-sense (hisG46) [2] bio, rfa, uvrB [2] Negative [18,27,28] Negative [24]
S. typhimurium TA1537c Mis-sense (hisC3076) [2] bio, rfa, uvrB [2] Positive Negative [24]
[18,20,21,28]
Negative [22,29]
E. coli WP2 uvrA pKM101 Mis-sense (trpE56) [30] pKM101 uvrA [30] N/A Positive [24]
Negative [25]
S. typhimurium TA97c Frameshift pKM101, rfa, uvrB [31] N/A N/A
(hisD6610) [31]
S. typhimurium TA97a Frameshift (hisD6610) [32] pKM101, rfa, uvrB [32] N/A Negative [22]
S. typhimurium TA102c Mis-sense (hisG428) [33] pKM10, pAQ1, rfa [33] Negative [20,27] Negative [22,24]
S. typhimurium TA104c Mis-sense (hisG428) [33] rfa,uvrB [33] N/A N/A
S. typhimurium TA1536 Frameshift (hisC207) [2] bio, rfa, uvrB [2] Negative [29] N/A
S. typhimurium TA1538 Frameshift (hisD3052) [2,6] bio, rfa, uvrB [2,6] Positive [18,28,29] Negative [24]
S. typhimurium YG1021 Frameshift (hisD3052) [34] pKM101, pYG216, bio, rfa, uvrB Nitroreductase N/A Positive [24]
overproducing [34]
S. typhimurium YG1024 Frameshift (hisD3052) [35] pKM101, pYG219 bio, rfa, uvrB N/A Positive [24,25]
O-acetyltransferase-overproducing [35]
S. typhimurium YG1026 Mis-sense (hisG46) [34] pKM101,pYG2016, bio, rfa, uvrB N/A Positive [24]
Nitroreductase overproducing [34]
S. typhimurium YG1029 Mis-sense (hisG46) [35] pKM101, pYG219, bio, rfa, uvrB N/A Positive [24]
O-acetyltransferase-overproducing [35]
S. typhimurium YG1041 Frameshift (hisD3052) [36] pKM101, pYG233 bio, rfa, uvrB Nitroreductase Positive [19] Positive [24]
and O-acetyltransferase-overproducing [36]
S. typhimurium YG1042 Mis-sense (hisG46) [36] pKM101, pYG233 bio, rfa, uvrB Nitroreductase N/A Positive
and O-acetyltransferase-overproducing [36] [24,25]
N/A - no information on the strain response to cigarette smoke or smoke fractions available. a Particulate phase generated using filter pad or aqueous particulate trapping techniques. b Whole smoke generated using smoke aerosol generator. c Strains assessed in this study.
inexpensive, rapid, and easy to perform, and capable of providing consistent and reliable results. An advantage of the assay is that the response across a variety of different tester strains can provide information on the mutagenic mode of action [6].
The Ames assay has been recognised globally by scientific communities, government agencies and corporations, and is used as an initial screen and/or early development assay to determine the mutagenic potential of new chemicals and drugs. The associated value of the Ames assay was also increased when it was discovered that a positive response in the assay showed a high predictive correlation with in vivo rodent carcinogenicity [7-9]. Ames assay conduct parameters are covered in several international regulatory guidelines, including those by the Organisation for Economic Co-operation and Development (OECD guideline 471) [10] and the International Commission on Harmonization (ICH S2R1) [11].These guidelines ensure the uniformity and robustness of data submitted to regulatory agencies for registration or acceptance of many chemicals. Although these guidelines are appropriate and suitable for the testing of compounds as solutions or suspensions, modifications are required to enable the testing of gases, such as individual cigarette smoke toxicants, and complex aerosols including cigarette smoke [9,12,13]. As a result, there is currently a drive by investigators involved in tobacco-related research to ensure the toxicological assessment of cigarette smoke is based on the complete smoke aerosol, and not just a smoke sub-fraction [14-17]. Therefore, a modified Ames methodology for aerosol exposure would be beneficial, particularly to the tobacco industry. Assessment of the entire smoke aerosol is of particular interest as new modified combustible tobacco products and aerosol-based nicotine products become available and widely used.
Currently OECD guidelines [10] recommend a total of at least five bacterial strains for chemical assessment. The recommendations are for four strains of Salmonella typhimurium (S. typhimurium)
includingTA98,TA100,TA1535, and either TA1537,TA97 or TA97a, which between them detect frameshift and base-pair substitutions, with GC base pairs at the primary reversion site. A fifth strain of either S. typhimurium strain TA102 or an Escherichia coli (E. coli) strain (WP2 uvrA or WP2 uvrA (pKM101)) is recommended to detect mutagens that may not be captured by the previous four strains, such as certain hydrazines and oxidising mutagens.
The mutagenic potential of cigarette smoke has been extensively investigated in the Ames assay using a variety of tester strains. Table 1 demonstrates a cross section of the strains that have been most widely used to assess the mutagenic potential of cigarette smoke, either through particulate analysis using standard Ames plate-incorporation methodology, or via an air-agar interface (AAI) aerosol exposure approach, as investigated in this study.
As demonstrated in Table 1, mutagenicity data have been generated on both cigarette smoke total particulate material (TPM) and a whole smoke aerosol. However, aerosol studies are limited in number compared to TPM studies. This is partly because it is far more convenient and practical to assess the mutagenic activity of tobacco products using a TPM test compound compared to generating and exposing bacterial cultures to whole mainstream cigarette smoke, where modifications to the standard Ames assay are required. In addition, TPM exposure techniques have been utilised in vitro by the tobacco industry and others in tobacco-related research for the last 40 years and, as such, there is a great deal of historical data available that allows tobacco research to be put into perspective. Unfortunately, this is not the case for whole smoke exposure techniques, which are still relatively new and establishing their presence as a reliable methodology for mutagenicity assessment.
In one 2008 whole smoke study, Aufderheide and Gressmann [24] used the CULTEX® exposure system to assess the mutagenic potential of a mainstream smoke aerosol from Kentucky reference cigarettes using 13 different tester strains, thus significantly
Fig. 1. Previously published and remodelled data forTA98, TA100, YG1024, YG1042 and E. coli (WP2 uvrA pKM101). A statistically-significant positive response to 3R4F cigarette smoke, in the presence of S-9 metabolic activation, was observed in TA98, TA100, YG1024 and YG1042, and a negative response observed in E. coli [25] following 24 min exposure. Data presented as a function of deposited mass (|xg/cm2) obtained in situ of exposure.
increasing the available knowledge. A study conducted by Kilford et al., [25], using the Vitrocell® exposure system with tester strains TA98, TA100, YG1024, YG1042 and E. coli (WP2 uvrA (pKM101)), demonstrated comparable results to the Aufderheide and Gress-mann study [24]. Despite these studies, there are still gaps in information in a number of key strains evaluated for the assessment of mainstream cigarette smoke using a 35 mm spread-plate AAI version of the assay. For example, there is little information on the mutagenic potential of whole smoke using strains TA1535, TA1537 and TA102, and information does not exist for TA97, TA97a and TA104. In the context of a regulatory assessment, knowledge onTA97 (and/orTA97a) andTA102 are essential as these strains are recognised alternatives for TA1537 and E. coli (WP2 uvrA or WP2 uvrA (pKM101)), respectively.
This study investigates five strains of S. typhimurium, TA1535, TA1537, TA97, TA102 and TA104, and analyses the data in combination with that previously generated by Kilford et al. [25] (Fig. 1). Through literature review, previous knowledge and experimental work, these strains were considered for their suitability when combined with both a 35 mm spread-plate methodology and an aerosol-based whole smoke exposure system.
By combining data obtained in this study with previously published data on strains, TA98, TA100, YG1024, YG1042 and E. coli (WP2 uvrA pKM101), a more comprehensive overview and approach to whole smoke/aerosol testing was considered based on strain compatibility within a Vitrocell® whole smoke exposure system, whilst maintaining adherence to recognised regulatory guidelines.
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 International Organisation of Standardisation (ISO) 3402:1999 ("Tobacco and tobacco products - Atmosphere for conditioning and testing").
2.3. Bacterial strains and culture conditions
Bacterial strains TA1535 and TA1537 were obtained from the National Collection of Type Cultures (NCTC), United Kingdom. TA97 was obtained from Professor Ames, University of California, Berkeley, USA. TA104 was obtained from BioReliance Corporation, USA, and TA102 was obtained from Covance Laboratories Inc., USA.
Prior to optimisation of culture conditions, each strain was checked for strain characteristics and antibiotic resistance according to Maron and Ames and De Serres and Shelby [4,37]. 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 8h.
2.4. Vitrocell® VC 10 setup and whole smoke exposure
The Vitrocell® VC 10 Smoking Robot (Vitrocell® Systems, Waldkirch, Germany) serial number VC10/090610, was used to expose bacteria to mainstream cigarette smoke generated from 3R4F reference cigarettes. The VC 10 is a rotary-head smoking machine which has a single syringe that transfers cigarette smoke to an independent, continuous airflow dilution bar. Smoke dilution is achieved via turbulent mixing with diluting air in the dilution bar and different smoke exposure concentrations are achieved by increasing or decreasing the diluting airflow. In addition to the diluting airflow, a vacuum sub-samples smoke (via negative pressure) from the dilution bar into the module, which docks directly under the dilution bar. Diluting airflow rates within this system were maintained using mass flow controllers (Analyt-MTC GmbH, Mülheim, Germany). Cigarettes were smoked to the ISO smoking regime (one 35 mL puff per 60 s, over a 2 s duration), according to ISO 3308:2012 ("Routine analytical cigarette-smoking machine - Definitions and standard conditions"), with an 8 s exhaust in serial-asynchron mode. Triplicate bacterial plates were exposed in Vitrocell® AMES 4 stainless steel exposure modules. The trumpet height within the module was set to 2 mm above the agar surface. Diluting airflows of 1.0,4.0, 8.0 and 12.0L/min, with a fixed vacuum of 5.0mL/min/well, were assessed in this study. Modules were exposed to a total of 3 or 8 cigarettes smoked over 24 or 64 min, respectively.
2.5. Ames reverse mutation assay
The Ames assay used in this study is modified from the standard 85 mm methodology and is described in detail in Kilford et al. [25]. Briefly, whole smoke exposures were performed in the presence and absence of an exogenous metabolic activation system (Aroclor 1254-induced rat liver S-9 from male Sprague-Dawley rats) (MolTox®, Molecular Toxicology, Inc., USA), in a scaled-down 35 mm plate format (Grenier Bio-One). Approximately, 2 x 107 bacteria cells were mixed with 75 |L sodium phosphate buffer (pH 7.4) or a 10% S-9 mix, prepared according to Ames et al. [2] and complemented with 40 | g/mL histidine and 48.8 | g/mL biotin mix. The bacterial cell suspension was plated directly onto Vogel-Bonner agar using a spread plate technique and incubated at 37 °C until dry (~20 min) before transferring to Vitrocell® AMES 4 exposure modules for exposure at the AAI. Concurrent controls (air, untreated and positive) were included with each exposure. Air controls were exposed to a constant flow of synthetic air (0.2 L/min diluting air flow, 5.0mL/min/well vacuum flow rate). Untreated and positive controls were maintained at room temperature for the duration of the exposure. Following exposure, the plates were removed from the exposure modules, sealed, inverted and incubated at 37 °C in the dark for 3 days. The resulting colonies on the plates were counted to determine the number of bacterial revertants/plate.
Table 2
Spontaneous revertant numbers for the 85 and 35 mm plate-format in the absence of metabolic activation.
TA1535 TA1537 TA97 TA102 TA104
85 mm rangea 5-30 2-25 114-171d 192-344 210-436e
Predicted 35 mm rangesb 1-5 0-4 19-29 33-58 42-87f
Observed 35 mm rangesc 0-5 0-5 6-25 26-66 17-45
a 99% reference range from 85 mm historical data.
b 99% reference ranges from laboratory historical data, scaled down from standard 85 mm plates. c 35 mm range generated from independent flowing air exposures.
d 99% confidence range based on a group mean of 4 plates, as no laboratory historical reference range exists for this strain. e No laboratory historical data available for the 85 mm assay. 85 mm range presented from Marnett et al. [41].
f Predicted scaled-down 35 mm ranges presented are scaled down from minimum and maximum values reported by Marnett et al. [41].
2.6. Data evaluation and acceptance criteria
Plates were scored using an automated colony counter (Sorcerer Image Analyser, Perceptive Instruments, Haverhill, UK) and the background lawn inspected for signs of toxicity, defined by a thinning of the background bacterial lawn or a marked reduction in revertant numbers. Responses with positive control chemicals were compared with laboratory historical observed ranges from data generated using this methodology. These ranges were comparable with historical control ranges held at Covance laboratories (Harrogate, UK) for the standard 85 mm plate assay, when these ranges are scaled down by a factor of approximately 5.9 for the reduced area on a 35 mm plate. Observed ranges of spontaneous revertant numbers for the 35 mm spread-plate methodology were also generated for all air and untreated control treatments. These ranges were comparable with historical control ranges held at Covance laboratories (Harrogate, UK), when scaled-down for the 35 mm plates, or published data where a historical control range was not available (i.e. for TA104). Data were evaluated using both fold increase in revertant numbers, over the concurrent air control plate counts, and statistically using Dunnett's test. For an increase in revertant numbers to be considered as a clear muta-genic response, the increases were required to be at least 2-fold (for strains TA97, TA102 and TA104) or 3-fold (with a minimum of 10 induced revertants/plate for strain TA1535) greater than the concurrent air control, statistically significant at the 1% level (p < 0.01) using Dunnett's test, and both concentration-related and reproducible over two or more independent experiments. For positive responding strains, 6 independent experiments were conducted, and for negative responding strains 2 independent experiments were conducted. Negative or equivocally-responding strains were further assessed using an extended, 64min exposure.
2.7. Measuring dose
A quartz crystal microbalance (QCM) (Vitrocell® Systems, Waldkirch, Germany) was included in situ of exposure at position 4 within the Ames module, distal to the smoke inlet, as previously demonstrated [38-40]. QCMs provide a gravimetric measure of deposited mass, providing a valuable quality control (QC) measure of smoke exposure within and between runs, and allows data to be presented as a function of deposited mass (^g/cm2).
3. Results
3.1. Culture optimisation
To ensure bacteria were in an actively dividing state when plated, and an appropriate cell culture density was achieved, growth curve assessments were performed on bacterial cultures of all strains by measuring optical density and plating for viable colonies every hour for up to 12 h of incubation at 37 °C. These assessments identified an optimal incubation time of 8 h to produce bacterial cell cultures that were in the late log phase of growth. Further assessments using positive controls demonstrated that the plating procedure and subsequent plate drying did not adversely affect the bacteria, and that bacteria were responsive and rapidly dividing. Prior to smoke exposure and assessment of smoke mutagenicity, all strains were assessed for their compatibility with the 35 mm scaled-down spread-plate methodology. Table 2 shows the spontaneous revertant numbers for each of the five strains assessed.
Data confirmed that strains TA97, TA102 and TA104 were compatible with this 35 mm scaled-down approach, with spontaneous revertant numbers of 6-25, 26-66, 17-45 compared to predicted 35 mm ranges of 19-29, 33-58, 42-87, respectively. However, strains TA1535 and TA1537 demonstrated low spontaneous rever-tant numbers, in-line with the predicted ranges based on standard 85 mm format. For example, TA1535 and TA1537 both produced 0-5 spontaneous revertants/plate, which were comparable to predicted 35 mm ranges (based on scaled down 85 mm historical values) of 1-5 and 0-4 revertants/plate, respectively. As a result of the low spontaneous revertant numbers, both TA1535 and TA1537 were considered sub-optimal for use with the 35 mm scaled-down plate format. The use of strains that provide such low spontaneous revertant counts are potentially problematic, due to the likelihood of high degrees of biological variability (in terms of fold increases over the air control counts), very small increases in revertant numbers potentially meeting the criteria for a positive response and zero plate counts frequently occurring. Because of these factors, TA97 was employed instead ofTA1537, as this strain provides notably higher spontaneous revertant counts, and is cited as an acceptable alternative to strain TA1537 in regulatory guidelines [10,11]. As no such alternative strain has been identified, TA1535 was progressed to whole smoke exposure along with TA97, TA102
Table 3
Positive control concentrations used for all strains in the presence and absence ofS-9 metabolic activation.
Bacterial strain Positive control concentration per plate (observed revertant numbers/plate)
Without S-9 activation With S-9 activation
TA1535 NaN3 0.8 |xg/plate (32-85) AAN 0.8 |xg/plate (4-25)
TA97 ICR-191 0.3 ^g/plate (74-108) AAN 0.8 ^g/plate (73-176)
TA102 MMC1.0 ^g/plate (77-183) AAN 3.2 ^g/plate (85-234)
TA104 2NF 6.4 |xg/plate (31-55) and7.5 |xg/plate (31-65) AAN 0.8 ^g/plate (102-223)
Abbreviations: NaN3 - sodium azide; AAN - 2-aminoanthracene; MMC- mitomycin C; 2NF- 2-nitroflourene; ICR-191 - 6-chloro- 9-[3-(2-chloroethylamino)propylamino] -
2- methoxyacridine dihydrochloride.
Fig. 2. Average mean revertants resulting from exposure to diluted mainstream cigarette smoke (A, 24 min and B, 64 min) generated from 3R4F reference cigarettes in S. typhimurium strains TA97, TA102, TA104 and TA1535 at diluting airflows of 12.0, 8.0, 4.0 and 1.0L/min, with a constant vacuum of 5.0 mL/min/well, in the presence of S-9. All data are plotted against concurrent air controls.
and TA104. Table 3 details the positive control chemicals, concentrations used (|ig/plate) and the observed responses as revertants per plate. All positive control treatments resulted in at least a two-fold increase in revertant colonies per plate over the concurrent air control counts. Positive controls were selected based on chemicals obtained from literature. For each strain, independent chemicals and dose ranges were investigated, demonstrating the responsiveness of these strains in the context of historical data and literature. Gaseous exposure systems exist [12] and could have been employed in this setup to show positive control responses. However, these systems are logistically challenging to employ with numerous EH&S issues. Furthermore, the responses in these systems are relatively uncharacterised and difficult to interpret in the context of the surrounding literature. For these reasons, simple positive control chemicals were employed to contextualise the response, as Table 3 demonstrates.
Positive, untreated and air controls were included concurrently alongside each exposure. The data collected for positive, air and untreated controls in each experiment were comparable to the scaled-down historical control ranges for the standard plate incorporation assay (data held at Covance Laboratories Ltd.) and/or the observed ranges generated using the 35 mm spread-plate methodology. The data obtained showed that there were no significant differences in the range of spontaneous revertant numbers on untreated plates compared to flowing air-exposed plates, confirming that the exposure technology does not have any effect on the spontaneous revertant numbers.
3.2. Assessing the mutagenicity of mainstream cigarette smoke
Four strains of bacteria (TA1535, TA97, TA102 andTA104) were exposed to diluted 3R4F mainstream cigarette smoke using an exposure time of 24 min (3 cigarettes, at 8 puffs/cig) and 64 min (8 cigarettes, at 8 puffs/cig). On each occasion, different concentrations of smoke were achieved using varying flow rates of diluting air (1.0, 4.0, 8.0 and 12.0 L/min). For all strains, cigarette smoke mutagenicity was assessed both in the presence and absence of S-9 metabolic activation. In the absence of metabolic activation no marked increases in revertant numbers were observed above that of the air or untreated controls for any strain.
In the presence of metabolic activation, positive responses were only observed in one strain, TA104 (Fig. 2). Statistically significant increases in revertant numbers were observed in 5 of the 6 independent experiments, when using the Dunnett's test at the 1% level (p < 0.01), following 24min exposures at 1.0L/min smoke
dilution. For the experiment that did not meet the 1% significance level acceptance criteria, it was analysed at the 5% level (p < 0.05) and found to be statistically different. However, a 2-fold increase over the controls was only observed in 1 independent experiment, with fold increases over controls of 2.0, 1.9, 1.8, 1.5, 1.5 and 1.7. Therefore, the response in TA104 was considered to be weak using a 24 min exposure. As a result of the weak response observed, TA104 was further assessed using a 64 min exposure. Following this extended exposure period, three out of three experiments resulted in fold increases of two-fold or above (2.0, 2.0 and 2.2), with the maximum smoke exposure concentration (1.0 L/min diluting airflow). Two out of the three independent experiments also met the 1% significance level acceptance criteria when analysed using Dunnett's test. The third experiment was found to be statistically different following assessment at the 5% level (p < 0.05) using the Dunnett's test. In strains TA1535, TA97 and TA102, no marked increases in revertant numbers were observed above the air control for either the 24 or 64 min exposures in the presence or absence of S-9. Given the revised acceptance criteria, TA1535 was considered non-responsive, as it failed to meet the induced revertants per plate criteria at the 24 and 64 min exposures, even though, 5-fold and 3fold increases in revertant numbers, over controls, were observed following 24 and 64 min exposures. No evidence of toxicity was observed in any strains throughout the study.
A QCM was included in all whole smoke exposures in order to quantify particulate deposition within the module at the exposure interface. Data provided by the QCMs were used as a QC check to monitor exposure conditions and provide a valuable gravimetric measure of particulate exposure expressed in deposited particle mass per surface area (^g/cm2). Following all exposures, the mean numbers of revertants were plotted against the mean deposited mass (Fig. 3).
Table 4 shows all data obtained in the presence of metabolic activation. Average mean revertants (MR) with standard deviation (SD), fold increase (FI) and recorded deposited mass (DM), following 24 and 64 min exposure to mainstream cigarette smoke. An asterix (*) indicates where statistically significant increases in revertant numbers were observed when analysed at the 1% level using Dunnett's test (p < 0.01).
4. Discussion
There is clear regulatory guidance on the Ames assay for in vitro testing of pharmaceuticals and chemicals governed by International Regulations and Guidelines [10]. These guidelines
Fig. 3. Average mean revertants resulting from exposure to diluted mainstream cigarette smoke generated (A, 24 min and B, 64 min) from 3R4F reference cigarettes in four strains of S. typhimurium (TA97, TA102, TA104 and TA1535) at diluting airflows of 12.0, 8.0,4.0 and 1.0L/min, with a constant vacuum of 5.0mL/min/well, in the presence of S-9 metabolic activation. Data expressed as a function of mean deposited mass (|g/cm2) obtained in situ of exposure.
acknowledge that alternative procedures need to be used for the testing of certain compounds such as gases and volatile chemicals. However, these guidelines do not take into account aerosol exposure, nor indicate the nature that any alternative assessment procedures may take. Testing smoke aerosols in vitro is especially important when considering that there may be complex relationships between chemicals in each phase, semi-volatiles, short-lived products of combustion or unknown aerosol interactions within non-combustible products [20,42,43].
This study has investigated an Ames 35 mm spread-plate AAI methodology [24,25,44], for the testing of a mainstream cigarette smoke aerosol using the Vitrocell® VC 10 Smoking Robot. The study was further supplemented with an in situ gravimetric measure of particulate deposition (| g/cm2) using QCM technology. S. typhimurium strains TA1535, TA1537, TA97, TA102 and TA104 were investigated as these strains have either not been previously investigated, or information is limited on the responsiveness of these strains alongside whole smoke exposure systems. Preliminary assessment of the spontaneous revertant numbers identified
the low reversion rate of TA1535 and TA1537 when scaled-down to the 35 mm plate-format (Table 2). TA97 was used as an accepted alternative to TA1537 in whole smoke exposures. However, due to the lack of an appropriately cited TA1535 alternative, TA1535 was assessed with whole smoke in the presence and absence of S-9 metabolic activation. In order to help address some of these potential assessment issues, modified assessment criteria were employed. The results show that in the absence of metabolic activation, no increases in revertant numbers were observed (Table 3), despite appropriate positive control responses. In the presence of metabolic activation, a positive response was only observed in strain TA104 (Figs. 2 and 3). No marked increases above controls were observed at any dilutions tested in strains TA1535, TA97 and TA102 (Table 4).
Guidelines recommend a total of at least five bacterial strains be employed for chemical assessment [10]. Four strains of S. typhimurium (TA98; TA100; TA1535 andTA1537 orTA97 orTA97a), and either S. typhimurium strain TA102 or E. coli strain WP2 uvrA or WP2 uvrA (pKM101) together with strain WP2 or WP2 (pKM101),
Table 4
Whole smoke data in the presence of S-9 metabolic activation.
Strain
Airflow (L/min) 24 min exposure 64 min exposure
MR ± SD FI ± SD DM ± SD(|g/cm2) MR ± SD FI ± SD DM ± SD(|g/cm2)
Air control 0.93 ± 0.59 - - 0.63 ± 0.58 - -
12.0 1.68 ± 0.29 2.63 ± 1.82 0.01 ± 0.00 1.10 ± 0.17 2.10 ± 1.68 0.03 ± 0.04
8.0 1.58 ± 0.88 2.23 ± 1.95 0.09 ± 0.02 0.77 ± 0.68 0.83 ± 0.76 0.22 ± 0.05
4.0 1.58 ± 0.96 1.90 ± 0.76 0.61 ± 0.14 1.83 ± 0.42 3.20 ± 1.97 2.02 ± 0.28
1.0 2.18 ± 1.02 3.15 ± 2.15 3.54 ± 0.22 1.67 ± 0.65 2.77 ± 1.94 11.15 ± 0.79
Air control 16.53 ± 3.78 - - 18.15 ± 0.21 - -
12.0 17.16 ± 4.70 1.04 ± 0.17 0.01 ± 0.16 18.5 ± 0.24 1.02 ± 0.00 0.10 ± 0.00
8.0 16.18 ± 3.61 0.98 ± 0.17 0.05 ± 0.06 22.17 ± 1.65 1.22 ± 0.11 0.21 ± 0.13
4.0 20.08 ± 3.00 1.25 ± 0.32 0.57 ± 0.14 21.67 ± 3.77 1.19 ± 0.22 1.27 ± 0.15
1.0 19.00 ± 6.16 1.14 ± 0.35 3.31 ± 0.29 24.17 ± 4.01 1.33 ± 0.24 6.13 ± 0.20
Air control 42.91 ± 8.85 - - 32.85 ± 4.03 - -
12.0 45.67 ± 12.04 1.06 ± 0.16 0.06 ± 0.05 35.83 ± 2.59 1.09 ± 0.05 0.04 ± 0.02
8.0 38.38 ± 8.27 0.90 ± 0.07 0.10 ± 0.03 32.17 ± 6.84 0.97 ± 0.09 0.30 ± 0.07
4.0 35.75 ± 4.18 0.84 ± 0.11 0.58 ± 0.04 36.83 ± 12.02 1.11 ± 0.23 1.48 ± 0.06
1.0 48.13 ± 5.68 1.14 ± 0.12 3.10 ± 0.45 29.67 ± 3.30 0.92 ± 0.21 6.56 ± 0.17
Air control 33.12 ± 8.29 - - 34.56 ± 5.78 - -
12.0 34.28 ± 7.71 1.04 ± 0.10 0.05 ± 0.08 27.54 ± 1.19 0.80 ± 0.10 0.01 ± 0.05
8.0 37.0 ± 12.39 1.10 ± 0.10 0.02 ± 0.06 35.68 ± 6.98 1.04 ± 0.04 0.22 ± 0.11
4.0 38.94 ± 6.22 1.22 ± 0.27 0.52 ± 0.18 49.23 ± 3.66 1.44 ± 0.14 1.58 ± 0.26
1.0 56.33 ± 11.96 1.73 ± 0.20* 3.48 ± 0.32 71.32 ± 13.06 2.06 ± 0.10** 9.36 ± 0.61
TA1535
Abbreviations: MR - mean revertants; FI - fold increases; DM - deposited mass; SD - standard deviation. * Statistical significance observed at the 1% level (p < 0.01) in 5 out of the 6 independent experiments. ** Statistical significance observed at the 1% level (p < 0.01) in 2 out of the 3 independent experiments.
200.00 n 150.00
> 100.00
£ 50.00 0.00
f <T _______1
0.00 1.00 2.00 3.00
Deposited mass {pg/cm2)
—*— YG1024 --©- YG1042 --tu— TA100 —I— TA98
60.00 -
E 20.00
J-i 1 I ,
...... I 1 —- 1
-TA104
-TA1535
0.00 1.00 2.00 3.00
Deposited mass (pg/cm2)
60.00 50.00 i 40.00
1 30.00
| 20.00 10.00 0.00
--*---TA102
--A-- E. coli (WP2 uvrA pKM101)
0.00 1.00 2.00 3.00
Deposited mass {pg/cm2)
Fig. 4. Proposed strain testing approach - mean revertants resulting from exposure to 24 min diluted mainstream cigarette smoke generated from 3R4F reference cigarettes. Graph A shows, strains TA98 and TA100 and their derivatives, YG1024 and YG1042 [25]. Graph B shows, strains TA1535, TA97 and TA104. TA97 is an acceptable alternative for TA1537, which demonstrated extremely low spontaneous reversion numbers in this study. Graph C shows strains E. coli (WP2 uvrA pKM101) [25] andTA102, an accepted alternative to E. coli. TA104 is included as it is considered that it may prove useful in augmenting the regulatory approach.
to detect mutagens that may not be captured by the previous four strains. Over two studies (this and Kilford et al. [25]), a total of 10 Ames strains have been assessed for their compatibility with the reduced 35 mm spread-plate methodology using the Vitrocell® VC 10 exposure system at the AAI. Whole smoke tested positive in five strains (TA98, TA100, YG1024, YG1042 and TA104), and negative in four strains (TA97, TA102, E. coli (WP2 uvrA pKM101) and TA1535) following 24 and/or 64min exposure to 3R4F mainstream cigarette smoke in the presence of S-9 metabolic activation. TA1537 was deemed sub-optimal with this 35 mm format and was not assessed with whole smoke exposure. No marked increases in revertant numbers were observed in any strain tested in the absence of S-9. Our data suggests that using this methodology, a mutagenic event can be detected after a 24 min whole smoke exposure, which equates to only three reference (3R4F) cigarettes, smoked to 8 puffs/cigarette. By increasing the exposure time to 64 min (8 cigarettes), a more consistent response may be observed, however, no responses were observed after a 64 min exposure in any strain that was non-responsive at the 24 min time-point.
Based on these observations, a proposal can be made for a combination of strains to be used for the assessment of mainstream cigarette smoke aerosol in vitro which closely adheres to the requirements of regulatory guidelines (Fig. 4).
The introduction of the plasmid pKM101 into the tester strains has been reported to increase the susceptibility to certain chemical mutagens [46]. Those strains carrying the plasmid pKM101, like TA98 (frameshift mutation), TA100 (missense mutation) and their YG descendent strains (YG1024, YG1042) were all mutated by mainstream cigarette smoke, which supports the theory that strains carrying the pKM101 plasmid are more sensitive to chemical mutagens, certainly in the respect to the complex chemical composition of cigarette smoke. However, this was not true of E. coli (WP2 uvrA pKM101) or TA102 which were not mutated under any of the experimental conditions tested, and have been reported to be sen-
sitive to hydrazines, oxidising mutagens and cross-linking agents. There is little or no other evidence of E. coli (WP2 uvrA pKM101) being used to assess any fraction of cigarette smoke and whole smoke has only once shown a weakly positive result [24] (Table 1). TA102 has been assessed previously in both TPM and whole smoke studies [20,22,24,27] and has been found to be unequivocally negative to cigarette smoke, which is in accordance with the findings of this study.
The introduction of additional plasmids resulted in derivatives of TA98 and TA100. YG1024 is a derivative of strain TA98 which over-expresses an O-acetyl-transferase gene that confers high sensitivity to the mutagenic action of nitroarenes and aromatic amines [34-36]. YG1042, which is a derivative of strain TA100 which overexpresses nitroreductase and shows a higher sensitivity towards the mutagenic activity of some nitro-aromatic compounds [36]. Both strains showed higher sensitivity to mainstream cigarette smoke compared to their parent strains [25], suggesting these may be more suitable for the analysis of the mutagenic potential of cigarette smoke. The observations in this study partially confirm the findings presented by Aufderheide and Gressmann [24], however, Aufderheide and Gressmann did not observe the same sensitivity difference in YG1042 compared to its parent strain, as presented here. Irrespective of this, it still demonstrates the potential advantage of using tester strains that are more sensitive to certain mutagens present in mainstream cigarette smoke, and is consistent with their use in related assays. For example, strain YG1024 is frequently used in urine mutagenicity experiments, as this strain has previously been shown to be more sensitive in detecting mutagenicity in human urine, caused by cigarette smoking, than its parent strain, TA98 [45].
TA102 and TA104 have been reported to be more sensitive than TA100 to substances such as oxidative mutagens, including peroxides, hydroperoxides, free radical generators, unsaturated aldehydes, carbonyl compounds and bleomycin [4,33,46,47].ln our
Table 5
Proposed testing strategy for tobacco-related products.
Bacterial strain TPM Traditional Modified
assessment3 combustible combustible product
productbc or alternative
tobacco categoryb,d,e
S. typhimurium X X X
S. typhimurium X X X
S. typhimurium X Xf Xf
TA1535
S. typhimurium Xg
TA1537
E. coli WP2 uvrA X X X
pKM101 or S.
typhimurium
S. typhimurium Xg X X
S. typhimurium X
S. typhimurium X
YG1024
S. typhimurium X
YG1042
a Particulate exposure using standard, 85 mm, Ames methodology. b Air-agar interface aerosol exposure using 35 mm spread-plate methodology. c Traditional cigarette-format combustible products.
d Modified cigarette-format combustible products or alternative tobacco aerosol products.
e Testing strategy dependent on product modifications and smoke/aerosol chemistries.
f Strain to be used with caution in scaled-down 35 mm spread-plate format, in conjunction with revised evaluation criteria recommended in this study. g Either strain TA1537 orTA97 recommended foruse.
studies, TA102 was shown to be non-responsive to cigarette smoke both in the presence and absence of S-9 metabolic activation, under all conditions tested. TA100 and TA104 both demonstrated a weak positive response to whole smoke generated from 3R4F reference cigarette smoke. TA104 could be considered as an augmentation to the standard battery of strains, especially where cigarette smoke has been modified, or for the assessment of alternative smoke-products that fundamentally differ from traditional cigarettes. It may also be used for studies of chemicals specifically aimed at the hisG48 site, where it has been shown to be more sensitive than either TA100 orTA102 [46,47].
TA1535 has been reported as non-responsive in both TPM and previous whole smoke studies [18,20,24], and our findings support these observations. To our knowledge, TA97 has not been previously assessed in conjunction with whole mainstream cigarette smoke, and has proved unresponsive to cigarette smoke in this study. Although largely unresponsive, TA1535, TA1537 and TA97 may be still be important in the assessment of modified or novel tobacco products in the future. Their lack of responsiveness to cigarette smoke does not delimit their usefulness in maintaining a selection of strains with the ability to detect a wider range of mutagens.
Currently, there are no recommended guidelines for the testing of aerosols in the Ames assay and, given the emergence of new tobacco-related aerosol technologies and modified combustible products, there should be awareness that these new products may have significantly altered aerosol characteristics. As such, it is important to consider a testing strategy for these products that is aligned with the regulatory standards, whilst taking account of the potential difference in mutagenic activity of products using these technologies compared to traditional combustible products. Table 5 highlights how the strains discussed herein may be employed for the assessment of the mutagenic activity of new tobacco products.
Importantly, for new products, aerosol chemistries will define the chemicals and classes of compounds present and the quantity compared to a combustible product. This may ultimately drive and help define the most appropriate strains to use, but preliminary analysis suggests more strains should be employed where products are less understood (Table 5).
5. Conclusions
Five Ames strains, TA1535, TA1537, TA97, TA102 and TA104 were assessed in conjunction with a Vitrocell® VC 10 whole smoke aerosol exposure system to increase, support and supplement existing scientific data. Furthermore, the results from this study were compared against a previously conducted study [25] using Ames strains TA98, TA100, YG1024, YG1042 and E. coli WP2 uvrA pKM101. Based on the available literature and data, the following strains may be employed in a regulatory-style format for the assessment of the mutagenic potential of cigarette smoke or alternative aerosol-based tobacco products:
• TA98 and TA100 are important to maintain in a smoke aerosol testing strategy as there is a great deal of historical data, especially from a TPM perspective [18-20,26,27]. However, the increased sensitivity of YG1024 and YG1042 could prove useful when comparing between tobacco products, especially aerosol based.
• TA97 is a regulatory accepted alternative to TA1537, which was deemed sub-optimal with the 35 mm methodology adopted in this study due to low spontaneous revertant numbers. There is no recommended alternative to strainTA1535 and data analysis and conclusions based on such low spontaneous revertant numbers should be carefully considered, to avoid reporting a false-positive response. There are no such issues associated with the use ofTA97 in a 35 mm plate format.
• Either E. coli (WP2 uvrA pKM101) or TA102 as a recognised alternative could be employed for the assessment of mainstream cigarette smoke.
• Although TA104 is not part of a recognised regulatory testing strategy, it has been previously suggested that it could complement the standard in vitro Ames test battery [47,48]. The results from this study support the suggestion that TA104 could augment the traditional strains, depending on the test article. This is important where the test article delivers significantly different chemicals compared to a traditional combustible product, such as oxidative mutagens, including free radical generators and reactive oxygen species, unsaturated aldehydes or carbonyl compounds.
In conclusion, this study has increased the information available in the scientific domain regarding the mutagenicity of whole smoke aerosol in strains TA1535, TA97, TA102 and TA104. It has further compared observations to previous Ames data for strains TA98, TA100, YG1024, YG1042 and E. coli WP2 uvrA pKM101, and contextualised a testing strategy for mainstream cigarette smoke that is in alignment with current recommended protocols.
Declaration of interest
The authors report no conflicts of interest and are employees of British American Tobacco or Covance Laboratories Ltd. Covance Laboratories Ltd., Harrogate, UK, conducted all experimental work and were funded by British American Tobacco.
Author contributions
David Thorne, Joanne Kilford, Deborah Dillon and Clive Meredith designed the study. Joanne Kilford and Michael Hollings managed and conducted all experimental work. Joanne Kilford analysed the data. David Thorne drafted the manuscript. Annette Dalrymple, Mark Ballantyne, Clive Meredith and Deborah Dillon provided scientific support. All authors approved the final manuscript.
Acknowledgements
The authors would like to acknowledge Julie Clements for her scientific and technical advice and Laura Porter-Williams, Adam Seymour, Lindsey Zirins and Michelle Simpson for their technical assistance.
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