There is growing evidence that adverse effects of microcystin-LR (MC-LR) are closely
related to oxidative stress processes, free radicals and DNA damage, and involve major
gene transcript changes. This study, utilizing gene expression analysis and plasma
chemistries was the first to measure the effects of MC-LR in whitefish (Coregonus
lavaretus L.), a feasible organism for pollution monitoring in aquatic systems. Fish
were injected with different concentrations of MC-LR (0, 10 and 100 microg/kg of body
weight) and then sacrificed at either 0, 8, 24, 48 or 72 h later, and their liver
tissue were harvested for detailed investigation. Specifically, we were interested
whether MC-LR is capable of: (i) modulating expression of two genes, tumor suppressor
gene p53 and cdkn1a, p53 direct transcription target, and (ii) inducing apoptosis
in whitefish liver. To study these effects, we developed a real-time qPCR assays useful
for measuring both p53 and cdkn1a gene transcript levels in liver. To obtain necessary
information for the study, either full-length p53 cDNA of whitefish (Wf-p53) was determined,
using molecular cloning and rapid amplification of cDNA ends (RACE), or as for Wf-cdkn1a,
specific primers were designed based on highly conserved regions of cdkn1a in fish.
The Wf-p53 was found to share the same characteristics with a known p53 mRNA sequence
of other vertebrates. Whitefish p53 amino acid sequence showed a high degree of homology
with the sequences from fishes, amphibians, and mammals. The injection study showed
that MC-LR at a higher dose, i.e. 100 microg/kg body weight, up-regulated expression
of p53 and cdkn1a genes in whitefish liver, as reflected by the continuous increase
in their mRNA levels through the whole experiment. Furthermore, DNA fragmentation
was observed in liver cells of whitefish after 24h of exposure to MC-LR (100 microg/kg)
that suggests the possibility of apoptosis. Finally, the study confirmed previous
observations of severe injury of the liver and loss of normal organ functions as revealed
by elevated levels of blood AspAT, AlaAT, and hepatosomatic index (HSI).