| dc.description.abstract | Aquatic animals inhabit very unstable natural environments, and are also always in
danger of exposures to intermittent variable toxic man made chemical substances, which
often impair their normal tissue and cellular homeostasis. Cellular protein chaperones
otherwise known as Heat Shock Proteins (HSPs) have been described to play very
important roles in maintenance of various cellular homeostatic pathways. The aim of this
study was to establish and validate an in-vitro cellular bioassay test system for analysing
HSP70 expressions in Nile tilapia (Oreochromis niloticus) from Lake Victoria, a tropical
freshwater fish 'of great ecological and economical importance.
The suitability of the established in-vitro primary hepatocyte cultures from
Oreochromis niloticus for HSP70 protein analyses was evaluated by microscopy, flow
cytometry and western immunoblots followed by enhanced chemiluminescence, (ECL)
method. The effects of temperature and toxic concentrations of cadmium, copper and zinc
on in-vitro expressions of HSP70 were studied using one-dimensional SDS-PAGE
followed by western immunoblots and the proteins detected using ECL method. Inductions
of HSP70 in 0. niloticus using sub-lethal concentrations of cadmium and estimated 96-h
LCso values for p-DDE and P-HCH in tilapia were studied using Quantitative reverse
transcriptase coupled with polymerase chain reaction (RT-PCR).
The results obtained using light and electron microscopy followed with single-cell
flow cytometric DNA counts showed that nearly 90% of the hepatocytes seeded at a
density of 2.5Xl OS cells per well did not significantly proliferate (p < 0.05) but remained
viable for at least three weeks with fetal calf serum (FCS) supplement, which also resulted
into increased culture longevity. Mixed primary monolayer/aggregate hepatocyte cultures
achieved by 10'% FCS supplement expressed consistent levels of albumin, a liver-specific
protein and basal HSP70. However, primary mono layers cultured without FCS supplement
exhibited limited longevity with declining albumin and HSP70 protein expressions.
Western immunoblot analyses of the effects of heat shock on the in-vitro cultures
showed a transient but increased accumulation ofHSP70 to 200% compared to controls set
at 100% eight hours post treatment (p < 0.05). Cadmium ions (Cd2+) induced increased
expression of HSP70 to 170% at concentrations of between 20-30 11M (p < 0.05).
However, copper and zinc ions only showed an increased induction of HSP70 at a higher
concentration (100 11M), where Cd2
+ exhibited toxic effects. Quantitative RT -PCR analyses
of liver HSP70 mRNA expressions from cadmium, p-DDE and B-HCH exposed fish
confirmed the results of the in-vitro western immunoblots. However, only inductions by
cadmium resulted into statistically increased expressions of HSP70 mRNA (p < 0.05).
The results of these studies revealed that mixed in-vitro hepatocyte primary cultures
are a good bioassay model system for studying cellular toxicity. However, the use of
HSP70 alone as a cellular biomarker of toxicity in wild tilapia populations may be
misleading due to the presence of high basal levels of HSP70. Furthermore, HSP70
induction appeared to be dependent upon the toxic substance and concentration used. The
degree of HSP70 inductions appeared to be stress or-specific and concentration dependent
as observed inthe transient induction of the heat shock-induced synthesis. The different
HSP70 isoforms that showed enhanced inductions upon stress exposure resembled the HSP
pattern that was characteristic for induced exposure effect and not for the observed initial
heat shock expressions. Such results support the hypothesis that heat shock and low
concentrations of toxic chemical compounds that induce increased expressions of protein
chaperones may, under certain conditions, have beneficial effects related to a stimulation
of endogenous cytoprotective mechanisms that act to restore vital cellular homeostasis | en |
