Vinyl chloride exacerbates liver damage caused by other factors such as diet at concentrations below the current safety restrictions. So, the use parameters for average populations must be reassessed. The main advantage of this technique is that it uses a chronic, non-invasive inhalation protocol that closely mimics human exposure while causing minimal stress to the animal.
Demonstrating the procedure will be Anna Lang, a post-doc, and Regina Schnegelberger, a graduate student, both from my laboratory. One week prior to the start of the inhalation experiments, weigh all of the experimental animals and weigh the amount of low or high-fat diet food to be given per cage. Provide food and water ad libitum, replacing the regular chow for each exposure group with low or high-fat diet chow, as appropriate.
Weigh the remaining food at the end of each feeding day and monitor the mice throughout the experiment to ensure that the health of the animals is maintained. To set up the vinyl chloride inhalation exposure system, ensure that the diluent air in both the experimental and control chambers is high-efficiency particulate air and activated carbon filtered, dried, and pressure-regulated before entering the respective flow measurement devices. Confirm that the temperature and relative humidity are monitored from within the experimental and control chambers, that the chamber exhaust is passed through a HEPA filter, a carbon dioxide probe, and an activated carbon filter, before entering the exhaust area of the chemical hood to ensure that the mice are receiving acceptable ventilation.
The custom software can be used to change, monitor, and record the environmental variables of interest during the inhalation exposures. To prepare the chamber for an inhalation exposure, start by turning off all the air flows in the experimental and control chambers. Open each chamber door and place absorbent bedding material, absorbent side up, on top of the excreta pan.
Then, wet the absorbent material to provide a comfortable humidity level of about 40 to 60%relative humidity throughout the exposure period and set the desired exposure level of vinyl chloride in the experimental chamber. When the chambers are ready, transfer the mice into individual cages within the inhalation chamber cage racks, randomizing each mouse's placement within the racks daily to ensure that each mouse is exposed homogeneously within the chambers. Then, place each cage rack into its respective chamber and close the chamber doors.
To conduct an inhalation exposure experiment, confirm that the valve for the vinyl chloride gas tank is in the open position and set the diluent flow for the experimental chamber to 25 liters per minute. Confirm that the rotameter on the control chamber is set to 25 liters per minute and check that all of the sensors are working correctly and displaying the expected results in both the experimental and control chambers. Throughout the exposure, monitor and record the exposure time, diluent flow, vinyl chloride flow, temperature, humidity, chamber pressure, carbon dioxide level, and theoretical vinyl chloride concentration in the experimental chamber.
Confirm that the temperature and humidity for the control chamber are also displayed, graphed, and recorded. About halfway through each exposure, break the glass tips on a vinyl chloride detector tube and a pretreat tube and attach the flow out end of the vinyl chloride detector tube to the detector tube pump. Use a short piece of tubing to attach the flow in end of the vinyl chloride detector tube to the flow out end of the pretreat tube and attach a short piece of tubing to the flow in end of the pretreat.
Remove a plug from one of the sampling ports near the breathing zone and attach the tubing from the flow in end of the pretreat tube to the sampling port. From the full in position, extend the handle on the piston of the detector tube pump to the full out position to pull 100 milliliters of sampled gas from the chamber into the vinyl chloride detector tube. After 90 seconds, push the handle back in.
After the last gas sample has been obtained, remove the tube from the sampling port of the chamber and reinsert the plug into the port. Inspect the color change in the vinyl chloride detector tube to ascertain the vinyl chloride concentration within the chamber and record the detector tube reading for comparison to the theoretical value. If any problems occur during the exposure, set the vinyl chloride flow to zero and increase the diluent flow to its maximum value to quickly purge the chamber.
Once the exposure duration has been reached, the software will automatically turn off the vinyl chloride flow. The 15 minute safety timer will then begin to count down the duration for the experimental chamber to clear the vinyl chloride. Once it is safe to remove the animals, click ok.
At the end of the exposure period, turn the vinyl chloride gas tank valve stopcock to the closed position and turn off all the air flows in the exposure chamber. Turn the rotameter until no air is flowing through the control chamber and remove the door from each chamber. Remove the cage racks from the chambers and transfer the mice back to their home cages under a laminar flow hood.
Then, dispose of any waste from the excreta pan according to the institutional health and safety regulations and clean the chamber doors, pan, exposure racks, and chambers. Here, the weekly body weight and biweekly food consumption of a 12 week experiment are shown. All of the mice gained weight throughout the course of this study.
However, while the mice in the high-fat diet groups gained more weight compared to the mice in the low-fat diet groups, the mice exposed to vinyl chloride did not gain more weight than the mice in the respective control groups. The food consumption also did not differ between groups. In these representatives photomicrographs of H&E stained liver sections, vinyl chloride caused no overt pathologic changes in the low-fat diet group.
High-fat diet feeding significantly increased steatosis, was further exacerbated by vinyl chloride exposure. Moreover, vinyl chloride exposure in the high-fat diet group resulted in some inflammatory foci. In the low-fat diet animals, vinyl chloride did not increase transaminase levels, while the high-fat diet alone slightly increased transaminase expression, with a significant enhancement of this effect observed in the high-fat diet group exposed to vinyl chloride.
Further, the high-fat diet significantly increased the liver to body weight ratios, although vinyl chloride did not significantly impact this effect. When implementing this protocol, always take care that the proper safety precautions are taken so that inadvertent exposure to vinyl chloride by humans is avoided. This procedure can answer questions about short-term, long-term, and cumulative effects of vinyl chloride exposure impact on animal health, welfare, and organ damage.
These studies impact the field of our environmental and occupational risk assessment. For example, our group now studies the mechanistic interactions between vinyl chloride and diet.
