A Thesis submitted to the Faculty of Medicine,
in fulfilment of the Requirements for the Degree of
Doctor of Philosophy / Numerous studies have assessed the effects of single
fatty acids on various aspects of lipid metabolism,
particularly cancer. Established cell lines have largely
been used for this purpose. The choice of control cells.
however, has often been inappropriate. There is also a
surprising lack of knowledge of the effects of fatty
acids in the "real world", in which normal cells in vivo
are presented with mixtures of dietary fatty acids.
Before transformed cells can be used as models of
disease states, it is essential to fully understand
fatty acid metabolism in normal (control) cells. Only
then can experimental findings be extrapolated to the
clinical situation with some certainty. This thesis has
therefore, assessed the effects of exogenous fatty acid
mixtures on the growth/viability of normal mammalian
tissues all cultured under standard conditions, and
attempted to elucidate the mechanisms underlying such
effects.
As different mammalian species exhibit different fatty
acyl desaturase capabilities, cells from three species
were chosen, viz. rat, Man and cat, with desaturase
capability decreasing with species, respectively. A wide
range of different cell types from each species were
studied due to the known differences in their lipid
profiles and metabolism. The cells studied in the rat
and cat included those derived from the cerebral cortex.
white adipose tissue, skin, lung, skeletal muscle and
aortic endothelium, while the human cell
obtainable included those derived from skin,
types
skeletal
muscle and lung. Erythrocytes and lymphocytes were
studied in all 3 species.
'Each of the cultured normal mammalian tissues studied
were shown to exhibit different proliferation rates.
Cultured cells were dosed with fatty acid mixtures
mimicking the fatty acid composition of dietary oils;
these mixtures were termed 'pesudo-oils", were bound to
bovine serum albumin as carrier, dosed at concentrations
ranging from 0 to 100mg/l, and cultures incubated for 48
hours. Viable cell numbers were assessed and related to
control cell numbers at the time of dosing. This was
termed the 'cytostatic number", and its implications
were discussed.
pseudo-Oils modulated cell viability, but the extent
thereof varied considerably in magnitude with pseudo-oil
and concentration dosed. Furthermore, differences in
cell viability were shown both between tissues from a
particular species, as ........ between identical
cultured tissues derived from different species. In
general, pseudo-oil supplementation of cultures induced
an overall concentration dependant growth limiting
and/or cytotoxic effect. Proliferation of certain cell
types were, however, stimulated with some pseudo-oils,
particularly at low to intermediate concontrations in
the range dosed. Differences in cell viability were also
related to the degree of pseudo-oii unsaturation; the
two most saturated oils, meat and coconut, were in
general, least effective in limiting and/or promoting
cell viability, while the effects induced with pseuciooils
rich in polyenoic fatty acids were more marked. The
possibility that the cell viability changes induced were
related to albumin as fatty acid carrier, or fetal calf
serum as a supplement in the incubation medium, were
investigated, and the possibility discounted. The use of
pseudo-oils rather than single fatty acids, however~
warrants consideration of fatty acid synergism and
antagonism.
To establish the possible mechanism(s) whereby the
dosed pseudo-oils inf!uenced cell viability, various
cell parameters were subsequently examined. These
included total protein, the incorporation, desaturation
and elongation of both pseudo-oil, and single C18, fatty
acids, as well as the production of lipoperoxides and
prostanoids.
total cellular protein concentrations varied both
between tissues and species, and reflected changes in
cell number in dosed cells However, pseudo-oils were
also shown to modulate absolute protein synthesis in
nucleated mammalian cells, primarily by enhancing such.
It was postulated that this related to the induction of
lipid merabolising enzymes.
Variations in cellular fatty acid composition were found
both between the mammalian tissues and species studied.
Evidence to support the capability of cultured mammalian
cells to incorporate exogenous fatty acids was shoun.
The extent of incorporationr however, varied with cell
type, species and fatty acid structure. Desaturase
cascade enzyme capability was also assessed by
comp~rison o~ dosed cell FA profiles witn that of
controls. Once again, desatwration and elongation
capability varied with different cell types within a
particular species, independent of cell proliferation
rates, but was generally greatest in dividing rat, lower
in human, and least in cat, tissues. In all three
species, however, erythrocytes and lymphocytes failed to
efficiently perform these reactions. This correlated
with the lower threshold for Cytotoxicity in lymphocytes
and erythroctyes than in dividing cells, although
desaturation capability did not correlate directly with
proliferation rates in growing cells. It was nevertheless
clearly shown that desaturase cascade enzyme
expression with pseudo-oil dosage was more limited than,
and could not be fully predicted by, the results
obtained utilising single FA's. This phenomenon was
related, to synergism and antagonism between pseudo-oil
FA's.
Cultured mammalian tissues were found to vary in their
capability to form both lipoperoxides and eicoeanoide ,
Pico-molar amounts of total eicoesanoide were quantitated
compared
to nano-molar
lipid peroxide
amounts of lipid peroxides.
production was not directly
related to control cell proliferation rates or total
unsaturated FA levels. However, total molar eicosanoid
concentrations in control cells and the eicosanoid:MDA
ratio suggested a correlation with the desaturation
capabilities of the three species, decreasing in the
order rat
exhibited
> human > cat each cell type studied
a unique prostanoid profile, which was
modulated (enhanced~ suppressed or inhibited) to a
greater or lesser extent with pseudo-oil incubation.
However. no correlation between cell viability, the
degree of pseudo-oil unsaturation, or any other
biochemical parameter studied except pseudo-oil
concentration dosed, could be proven to relate to the
changes in prostanoid levels induced with pseudo-oil
supplementation. This implied that endogenously biosynthesised
prostanoida were not directly responsible
for' effects induced. On the other hand, lipoperoxide
production generally increased with pseudo-oil
concentration dosed and was greater wi th pseudo oils
rich in Polyenoic than moroenoic or saturlted FA's.
Furthermore lipoperoxide involvement in the modulation
mammalian cell proliferation was proposed,
however, was shown not to be the sole mechanism, and
the involvement of membrane structural changes, ego
fluidity was
The findings
indicated that the modulation of cell
proliferation by FA"s was multifactorial,
The variations found between rat, cat and human tissues
with regard to the parameters investigated indicated
that extrapolation of experimental results between
mammalian tissues and species, as well as the use of
cells from different tissues and particularly from
different species as controls for other cells, is
potentially misleading, and should be avoided if
reliable interpretation of results is desired. Further
more, we recommend fatty acid analysis of all culture
media prior to use, and appropriate supplementation with
polyenoic,
required.
or at least essential, fatty acids if
Since p-oil FA composition reflected that of dietary
oiIs, the data from this thesis serves as an in vitro
model and guide to how normal genetically entire
mammalian cells in vivo may respond when similar oils
form part of the dietary intake. / Andrew Chakane 2018
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/24757 |
| Date | January 1991 |
| Creators | Fubbs, Joanmariae Louise, Giangregorio, Giangregorio |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf, application/pdf, application/pdf |
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