| Summary: | Dietary and environmental hepatocarcinogens will be metabolized to active
compounds and it must be detoxified in order to maintain liver integrity. In this
study the feeding of nitrosamines and their effects on turnour marker enzymes
Alkaline Phosphatase (ALP), Gamma-Glutamyl Transpeptidase (GGT),
Glutathione S-transferase (GST) and Uridyl diphospho-glucuronosyl transferase
(UDPGT) were analyzed in mice liver.
The initial work involved homogenization of liver samples with different buffers
at various concentrations. Results with ALP and GGT shows highest specific
activities for liver samples extracted with 0.01M Tris-HC1 at pH 7.5. Further
work on the use of different solvents, surfactants and detergents to optimize the
extraction of alkaline phosphatase and gamma-glutamyl transpeptidase were
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conducted. The results obtained showed that 0.01M Tris-HC1 buffer at pH 7.5
alone is sufficient to extract these membrane bound enzymes.
Acute studies were conducted by feeding mice with 2-20% of LDS0 of NNitrosodimethylamine
(NDMA) and N-Nitrosodiethylamine (NDEA) and mice
were killed at 24th, 36', 4gth, 60' and 72"* hours and the liver ALP and GGT
were assayed for their activities. Mice fed with 5mg of NDMAlkg of body
weight dose for 36 hours showed highest and significant (p<0.05) activation of
liver ALP and GGT compared to respective controls suggesting that feeding of
NDMA had activated liver marker enzymes activities. The enzyme activities of
ALP and GGT for treated mice were 4.215 IUIg protein and 0.656 IUIg protein
respectively and in the control liver ALP activity were 1.084 IUIg protein and
GGT activity were 0.375 W/g protein.
Chronic toxicity study was conducted with oral feeding of 5mg NDMAkg of
body weight on weekly basis for 20 weeks. The control and treated mice were
sacrificed every fortnight. The severity of neoplasia was studied by histological
evaluations and the activity of ALP, GGT, GST and UDPGT were assayed.
Studies on these enzymes show significant elevation at (p<0.05) for ALP, GGT
and GST compared to respective controls. UDPGT does not show any changes
in control and treated mice. ALP and GST was significantly (p<0.05) elevated
compared to control at 2"*, 16& and 20' week and GGT was significantly higher
than control at week 8", loh, 1 6 a~nd 2 0 ~ T. he highest enzymes activities
PERPUsTAKAAN SULTAN ABOUL SAMAD
UlllVWSlTl PUTRA MALAYSIA
measured for the three enzymes were on the 20' week of experiment. The
activities of liver enzyme in treated mice were 5.63 IUIg protein, 1.55 IUlg
protein and 2.55 pnole/min/mg protein respectively for ALP, GGT and GST and
the activities in control mice during the same week was 1.27 IUIg protein for
ALP, 0.376 IUIg protein for GGT and 1.39 j~molelminlmg protein for GST.
Histological evaluations through Hematoxilin and Eosin (H&E) staining and
Transmission Electron Microscopy (TEM) obtained showed chronic ingestion
had caused loss of normal cell organization in liver. Observation with H&E
staining and TEM also showed the shrinking of nucleus, cellular and vacuolar
degeneration and paler hepatocytes. From 10' week onwards significant
@<0.05) increase in lesion score in liver compare to control liver was observed
in slides stained with H&E. The present result suggests even at low dose and at
weekly feeding to mice, NDMA is capable in elevating turnour marker enzymes
in liver and this compound also caused disruption to the normal cell organization
of the liver.
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