Identification of hepatic mitogenic and cytochrome P-450-inducing fractions of unleaded gasoline in B6C3F1 mice.

Unleaded gasoline (UG), a complex mixture of over 300 hydrocarbons, induced liver tumors selectively in female mice and exhibited liver tumor promoting activity. UG also induced cell proliferation and cytochrome P-450-related enzyme activities in mouse liver, properties commonly associated with liver tumor promoters. To determine if the mitogenic and/or cytochrome P-450-inducing properties of UG reside in individual fractions of UG, UG was separated into four fractions on the basis of boiling point (BP): fraction 1, BP < 66 degrees C; fraction 2, 66 degrees C < BP < 100 degrees C; fraction 3, 100 degrees C < BP < 132 degrees C; fraction 4, BP > 132 degrees C. Fractions 1 and 2 were combined to form "light UG" (BP < 100 degrees C), and fractions 3 and 4 were combined to form "heavy UG" (BP > 100 degrees C). Female B6C3F1 mice were implanted with osmotic pumps containing 5-bromo-2'-deoxyuridine (BrdU) on d 1, treated by intragastric intubation with corn oil or 3000 mg/kg/d of light, heavy, or whole UG on d 2-4, and euthanized on d 5. Pentoxyresorufin O-dealkylase (PROD) and ethoxyresorufin O-deethylase (EROD) activities were assayed in hepatic microsomes, and hepatocyte BrdU labeling index (LI) was determined in liver sections. Whole UG and heavy UG caused comparable increases in hepatic PROD and EROD activities and the hepatocyte LI. Light UG caused relatively small increases in hepatic PROD and EROD activities and did not increase the hepatocyte LI. When fractions 3 and 4 were tested separately in the above treatment protocol, both fractions strongly induced hepatic PROD and weakly induced hepatic EROD activities. However, only fraction 3 increased the hepatocyte LI. To isolate mitogenic components in fraction 3, equimolar doses of individual chemicals in fraction 3 were tested in the above treatment protocol. Toluene did not increase the hepatocyte LI, whereas 2,2,3-trimethylpentane (TMP), 2,2,4-TMP, and 2,3,4-TMP all dramatically increased the hepatocyte LI. Thus, while the hepatic cytochrome P-450-inducing activity of UG was concentrated in components of UG with BPs > 100 degrees C, this activity apparently resides in UG components with a wide range of BPs. The mitogenic activity of UG, in contrast, was highly concentrated in components of UG with BPs ranging from approximately 100 to 132 degrees C, and quite possibly in specific TMPs.