Histochemical studies of the effect of puromycin on the liver in the mouse
It is known that the liver is one of the important organs which is closely related to endocrine and exocrine functions, hemopoiesis, coagulation, phagocytosis, and detoxication. Important substances needed to carry on these functions, including that of protein synthesis, are constantly being produced and metabolized in the liver. This is a study of the effect of puromycin on hepatic function in mice.
Puromycin was extracted from Streptomyces albo-niger by Porter et al.(1952). Clinically, puromycin is one of the antibiotics which effectively controls Trypanosoma gambiense and Entamoeba histolytica infections ets. Also it causes a moderate regression of the tumor in disseminated or advanced neoplastic disease.
Nathans et al.(1963) reported that protein synthesis was inhibited by puromycin. Many researchers have confirmed this inhibition. Morris et al.(1963) suggested the mechanism of puromycin action as follows : the inhibitor substitutes for the next incoming amino acid at the growing point of the peptide chain (the carboxylend). Nathans (1964) also reported that puromycin inhibits synthesis by blocking the transfer of amino acids from amonoacyl-sRNA to the peptide chain. Recently Guidice et al. (1968), Cundliffe and McQuillen (1968), and Longnecker et al. (1968) also reported the inhibiting effect of puromycin on
Lieberman et al.(1963), Holland(1963), Sells(1968), reported that puromycin suppresses nucleic acid metabolism. Estensen and Baserga(1968) found that a chemical and radioautographic analysis of the small intestine of mice, injected with puromycin, revealed and immediate decrease of the precursor incorporation into DNA and protein. A delayed decrease of precusor incorporation into RNA was noted. In addition, such a decrease of precursor incorporation occurred only in the crypt cells, but was not found in the villi of the small intestine, the germinal centers of the spleen, and the liver. In contrast, there are some reports of chemical analysis of the effects puromycin on biosynthesis or enzyme activity; Griffin and Cox(1966) for alkaline phosphatase;
Weber and Singhal (1964) for glucose-6-phosphatase metabolism and acid phosphatase; and Conney and Gilman (1968) for microsomal TPNH-dependent enzymes. This present histochemical study observe the enzymatic changes of the liver in the puromycin treated mouse and confirms its biochemical suppression of protein
synthesis metabolism. The correlation of the puromycin action on protein synthesis with important hepatic enzymatic activities is also discussed.
Materials and Methods
Healthy adult male mice weighing an average of 20gm. were divided into two groups, the control and experimental. Food were given ad libitum. Puromycin dihydrochloride (Nutritional Biochemical Corp.) in aqueous solution was injected intraperitoneally. A single dose of 50mg./kg. body weight was given at 12-hr. intervals for 2, 4 and 7 days respectively.
On the day after last injection the animals were killed by spinal destruction, specimens of hepatic tissue were removed, and treated histochemically to demonstrate nucleic acids (methyl green-pyronin stain, Rosa, 1950), acid phosphatase (Eranko, 1952), alkaline phosphatase (Barker and Anderson, 1963), adenosine triphosphate and dihydronicotinamide adenine dinucleotide diaphorase (Wolfe and Cohen, 1963).
In addition, incubation without substrates was carried out as the contrast test, and all of these gave negative results.
Summary and Conclusions
The activities of acid phosphatase, alkaline phosphatase, adenosine triphosphatase, succinic dehydrogenase, dihydronicotinamide adenine dinucleotide phosphate and dihydronicotinamide adenine dinucleotide diaphorase, and the changes of nucleic acid in the liver of the mouse were histochemically observed in healthy male mice following injection of large doses of puromycin. The following results were obtained.
1. In the ribonucleic acid distribution : pyroninophilic substance began to decrease in the hepatic cells in the 2-day group. A considerable decrease was observed in the 4 and 7-day group. A gradual decrease of the pyroninophilic substances was observed in hepatic cells.
2. No remarkable or recognizable decrease of acid phosphatase activity was found in the 2 and 4-day group but enzyme activity was slightly reduced in the middle and peripheral zone of the hepatic lobule. In contrast with acid phosphatase, alkaline
phosphatase activity in the peripheral zone and bile canaliculi was reduced in the 4 and 7-day group.
3. The adenosine triphosphatase activity of the hepatic cells was decreased immediately. The sinusoids, central vein and interlobular vessels, which had strong enzymatic activity in the control, showed a slight decrease in the 4 and 7-day group.
4. With regard to the succinic dehydrogenase activity, in the control group the peripheral zone of the hepatic lobule was more active than the central zone, whereas in the 2-day group the peripheral zone of the hepatic lobule showed decrease in enzymatic activity and in the 4-and 7-day group both the middle and peripheral zone activity was decreased in the 4 and 7-day group.
5. In the dihydronicotinamide adenine dinucleotide phosphate diaphorase activity, the central and middle zone showed a remarkable decrease in the 4 and 7-day group. In the dihydronicotinamide adenine dinucleotide diaphorase, no recognizable enzymatic changes were found.
The above results showed that puromycin caused a decease in ribonucleic acid and enzymatic activites. It was suggested that such a suppression of hepatic enzymatic activities in the mouse coincides with the previously known biochemical findings, namely, that puromycin inhibited protein synthesis.