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Development of a BALB/c 3T3 neutral red uptake cytotoxicity test using a mainstream cigarette smoke exposure system.

Thorne D, Kilford J, Payne R, Haswell L, Dalrymple A, Meredith C, Dillon D - BMC Res Notes (2014)

Bottom Line: In addition, we also demonstrated significant dose-for-dose differences between mainstream cigarette smoke and the GVP fraction (P < 0.05).We have adapted the NRU methodology based on the ICCVAM protocol to capture the full interactions and complexities of tobacco smoke.This methodology could also be used to assess the performance of traditional cigarettes, blend and filter technologies, tobacco smoke fractions and individual test aerosols.

View Article: PubMed Central - HTML - PubMed

Affiliation: British American Tobacco, Group R&D, Southampton, Hampshire SO15 8TL, UK. David_Thorne@bat.com.

ABSTRACT

Background: Tobacco smoke toxicity has traditionally been assessed using the particulate fraction under submerged culture conditions which omits the vapour phase elements from any subsequent analysis. Therefore, methodologies that assess the full interactions and complexities of tobacco smoke are required. Here we describe the adaption of a modified BALB/c 3T3 neutral red uptake (NRU) cytotoxicity test methodology, which is based on the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) protocol for in vitro acute toxicity testing. The methodology described takes into account the synergies of both the particulate and vapour phase of tobacco smoke. This is of particular importance as both phases have been independently shown to induce in vitro cellular cytotoxicity.

Findings: The findings from this study indicate that mainstream tobacco smoke and the gas vapour phase (GVP), generated using the Vitrocell® VC 10 smoke exposure system, have distinct and significantly different toxicity profiles. Within the system tested, mainstream tobacco smoke produced a dilution IC50 (dilution (L/min) at which 50% cytotoxicity is observed) of 6.02 L/min, whereas the GVP produced a dilution IC50 of 3.20 L/min. In addition, we also demonstrated significant dose-for-dose differences between mainstream cigarette smoke and the GVP fraction (P < 0.05). This demonstrates the importance of testing the entire tobacco smoke aerosol and not just the particulate fraction, as has been the historical preference.

Conclusions: We have adapted the NRU methodology based on the ICCVAM protocol to capture the full interactions and complexities of tobacco smoke. This methodology could also be used to assess the performance of traditional cigarettes, blend and filter technologies, tobacco smoke fractions and individual test aerosols.

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Related in: MedlinePlus

A schematic representation of the Vitrocell® VC 10. [A] Computer, software controller, which determines machine settings and smoking parameters. [B] Smoking Robot carousel where cigarettes are loaded and smoked. For GVP studies a Cambridge filter pad was installed into the line between the smoking carousel and the piston, for removal of the particulate smoke fraction. [C] Piston and syringe, which draws and delivers cigarette smoke to the dilution system. [D] Dilution of whole smoke occurs in the dilution bar. [E] Smoke exposure module which maintains cells at the ALI. To measure particulate dose, a quartz crystal microbalance was incorporated into the chamber, as shown, in position 4.
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Figure 1: A schematic representation of the Vitrocell® VC 10. [A] Computer, software controller, which determines machine settings and smoking parameters. [B] Smoking Robot carousel where cigarettes are loaded and smoked. For GVP studies a Cambridge filter pad was installed into the line between the smoking carousel and the piston, for removal of the particulate smoke fraction. [C] Piston and syringe, which draws and delivers cigarette smoke to the dilution system. [D] Dilution of whole smoke occurs in the dilution bar. [E] Smoke exposure module which maintains cells at the ALI. To measure particulate dose, a quartz crystal microbalance was incorporated into the chamber, as shown, in position 4.

Mentions: A Vitrocell® VC 10 Smoking Robot (Serial Number - VC10/090610) and 6/4 CF stainless steel exposure modules (Vitrocell® Systems, Waldkirch, Germany) were used to generate, dilute and deliver cigarette smoke to BALB/c 3T3 cells maintained at the ALI. The VC 10 is a rotary style smoking machine which has a single syringe that transfers the tobacco smoke to an independent continuous flow dilution system. The Vitrocell® dilution system uses both airflow (L/min) and vacuum rate (mL/min) to define the exposure concentration. Smoke dilution is first achieved via turbulent mixing in the dilution bar and different smoke 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 system into the module, which docks directly under the flow dilution system (Figure 1). The flow rate of the vacuum dictates the flow rate over the cells and was therefore maintained at 5.0 mL/min/well for all treatments.


Development of a BALB/c 3T3 neutral red uptake cytotoxicity test using a mainstream cigarette smoke exposure system.

Thorne D, Kilford J, Payne R, Haswell L, Dalrymple A, Meredith C, Dillon D - BMC Res Notes (2014)

A schematic representation of the Vitrocell® VC 10. [A] Computer, software controller, which determines machine settings and smoking parameters. [B] Smoking Robot carousel where cigarettes are loaded and smoked. For GVP studies a Cambridge filter pad was installed into the line between the smoking carousel and the piston, for removal of the particulate smoke fraction. [C] Piston and syringe, which draws and delivers cigarette smoke to the dilution system. [D] Dilution of whole smoke occurs in the dilution bar. [E] Smoke exposure module which maintains cells at the ALI. To measure particulate dose, a quartz crystal microbalance was incorporated into the chamber, as shown, in position 4.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4067082&req=5

Figure 1: A schematic representation of the Vitrocell® VC 10. [A] Computer, software controller, which determines machine settings and smoking parameters. [B] Smoking Robot carousel where cigarettes are loaded and smoked. For GVP studies a Cambridge filter pad was installed into the line between the smoking carousel and the piston, for removal of the particulate smoke fraction. [C] Piston and syringe, which draws and delivers cigarette smoke to the dilution system. [D] Dilution of whole smoke occurs in the dilution bar. [E] Smoke exposure module which maintains cells at the ALI. To measure particulate dose, a quartz crystal microbalance was incorporated into the chamber, as shown, in position 4.
Mentions: A Vitrocell® VC 10 Smoking Robot (Serial Number - VC10/090610) and 6/4 CF stainless steel exposure modules (Vitrocell® Systems, Waldkirch, Germany) were used to generate, dilute and deliver cigarette smoke to BALB/c 3T3 cells maintained at the ALI. The VC 10 is a rotary style smoking machine which has a single syringe that transfers the tobacco smoke to an independent continuous flow dilution system. The Vitrocell® dilution system uses both airflow (L/min) and vacuum rate (mL/min) to define the exposure concentration. Smoke dilution is first achieved via turbulent mixing in the dilution bar and different smoke 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 system into the module, which docks directly under the flow dilution system (Figure 1). The flow rate of the vacuum dictates the flow rate over the cells and was therefore maintained at 5.0 mL/min/well for all treatments.

Bottom Line: In addition, we also demonstrated significant dose-for-dose differences between mainstream cigarette smoke and the GVP fraction (P < 0.05).We have adapted the NRU methodology based on the ICCVAM protocol to capture the full interactions and complexities of tobacco smoke.This methodology could also be used to assess the performance of traditional cigarettes, blend and filter technologies, tobacco smoke fractions and individual test aerosols.

View Article: PubMed Central - HTML - PubMed

Affiliation: British American Tobacco, Group R&D, Southampton, Hampshire SO15 8TL, UK. David_Thorne@bat.com.

ABSTRACT

Background: Tobacco smoke toxicity has traditionally been assessed using the particulate fraction under submerged culture conditions which omits the vapour phase elements from any subsequent analysis. Therefore, methodologies that assess the full interactions and complexities of tobacco smoke are required. Here we describe the adaption of a modified BALB/c 3T3 neutral red uptake (NRU) cytotoxicity test methodology, which is based on the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) protocol for in vitro acute toxicity testing. The methodology described takes into account the synergies of both the particulate and vapour phase of tobacco smoke. This is of particular importance as both phases have been independently shown to induce in vitro cellular cytotoxicity.

Findings: The findings from this study indicate that mainstream tobacco smoke and the gas vapour phase (GVP), generated using the Vitrocell® VC 10 smoke exposure system, have distinct and significantly different toxicity profiles. Within the system tested, mainstream tobacco smoke produced a dilution IC50 (dilution (L/min) at which 50% cytotoxicity is observed) of 6.02 L/min, whereas the GVP produced a dilution IC50 of 3.20 L/min. In addition, we also demonstrated significant dose-for-dose differences between mainstream cigarette smoke and the GVP fraction (P < 0.05). This demonstrates the importance of testing the entire tobacco smoke aerosol and not just the particulate fraction, as has been the historical preference.

Conclusions: We have adapted the NRU methodology based on the ICCVAM protocol to capture the full interactions and complexities of tobacco smoke. This methodology could also be used to assess the performance of traditional cigarettes, blend and filter technologies, tobacco smoke fractions and individual test aerosols.

Show MeSH
Related in: MedlinePlus