Ciguatera fish poisoning (CFP) is a foodborne illness caused by consumption
of a variety of reef fishes contaminated with a group of natural marine toxins called
ciguatoxins (CTXs). CTXs are a group of natural marine toxins consisting of more
than 20 congeners, of which 14 have been structurally characterized. CTXs are
extremely potent - the lethal potency of the most toxic CTX, Pacific-CTX-1 (PCTX-
1), is 100 times greater than that of tetrodotoxin. Toxicity is related to the
uncontrolled activation of voltage-sensitive sodium ion (Na+) channels by CTXs in
the membranes of nerve cells. Consequently, symptoms of ciguatera include
gastrointestinal, neurological and cardiovascular disorders such as abdominal pain,
vomiting, nausea, diarrhea, paraesthesia, reversal of hot and cold sensations,
numbness and tingling and perception of loose teeth. The safe level of exposure to PCTX-
1 equivalents in fish has been proposed as 0.01 ng/g of fish flesh based on
results from the mouse bioassay toxicity test and including an uncertainty factor of 10.
Outbreaks of CFP have had both public health and socioeconomic impacts for
more than a century. CFP is not limited to tropical regions, but also occurs in nonendemic
regions because of the expanding international trade in tropical fish species and the lack of practical and accurate testing methods for CTXs. There is currently no
approved treatment available for CFP, and therefore avoidance of consuming
ciguateric fish is the most practicable way to prevent CFP outbreaks. Both diagnosis
of CFP and detection of CTXs is challenging. Diagnostic methods are limited by the
difficulty in differentiating CFP symptoms from those of other types of seafood
poisoning; current methods are based on diagnosing the intoxication symptoms prior
to detection of CTXs in the fish remaining from the meal or in blood plasma of
patients.
The difficulties of CTX detection are due to the odorless and tasteless nature
of CTXs and the fact that they have no effect on the appearance of ciguateric fishes.
Moreover, the high potency of CTXs means that extremely small amounts of CTXs
can cause severe poisoning, intensifying the difficulties of detection. Therefore a fast,
highly specific and sensitive detection method is needed for discrimination of toxic
from non-toxic fish specimens for surveillance and public health purposes. The
identification of biomarker(s) related to CFP can support the development of a more
specific detection method, greater understanding of the mechanism(s) of intoxication,
and the resistance and biotransformation mechanisms that are present in ciguateric
fishes.
In this study, coral reef fish samples were collected from a ciguatera-endemic
area, the Republic of Kiribati. The ciguatoxicity of the collected fish samples was
assessed using the mouse neuroblastoma assay (MNA) using P-CTX-1 as the
standard after rapid extraction. The ciguatoxicities of each specimen were compared
and toxic and non-toxic individuals were selected for protein expression
investigation. Two-dimensional gel electrophoresis (2-DE) coupled with silver
staining was used to investigate the expression profiles and the protein spots of
interest were identified by matrix-assisted laser desorption/ionization time-of-flight
mass spectrometry (MALDI-ToF-MS) with Macsot database searching. The protein
expression profiles in grouper (Epinephelus coioides) GL-a liver cells and in
juvenile E. coioides exposed to P-CTX-1 were also compared. Stable isotope
labeling with amino acids in cell culture (SILAC) was used with a non-gel-based
proteomic approach and liquid chromatography-tandem mass spectrometry (LCMS/
MS) for performing differential display analysis in the GL-a cells, while 2-DE
was used for analysis of the juvenile groupers. Heavy [13C6 L-arginine (R6),2H4 LLysine
(K4) labled], medium [13C6
15N4 L-arginine (R10), 13C6 L-Lysine (K6)] and
light [normal L-arginine (R0) and L-lysine (K0)] amino acids were incorporated into
the cells during culturing prior to experimental exposure. The juvenile groupers
were fed with grouper larvae pre-injected with P-CTX-1. The proteins were extracted from the GL-a cells and juvenile groupers after exposure and digested for
analysis by nano flow liquid chromatography quadrupole time-of-flight MS (Nano-
LC-QToF-MS) and MALDI-ToF-MS, respectively.
Base on the MNA results, 156 fish specimens, making up of 91% of fish
samples, were ciguatoxic based on the 0.01 ng/g safety level. Among the
carnivorous fishes, most of the groupers (91%) and all of the moray eels were
ciguatoxic. The groupers C. argus and E. spilotoceps were found to contain high
levels of P-CTX-1 equivalents. The ciguatoxicity of liver was an average of nine
times more toxic than muscle (ranging from 4.09- to 15.43-fold) in six individual
Gymnothorax spp. These results show that these two groups of fish can be used for
protein expression screening to investigate CTX-related proteins.
Protein profiles of the livers of ciguatoxic and relatively non-toxic groupers, E.
spilotoceps, were filtered by selecting proteins with ≥ 3.0-fold change in expression
and a total of 35 spots were selected. Among these 35 spots, 23 spots were found to
be up-regulated compared to the relatively non-toxic sample (reference gel) and 12
protein spot were down-regulated. Several up-regulated proteins were identified,
including triosephosphate isomerase B, ferritin subunit and muscle fatty acid binding protein. The identified down-regulated protein potentially associated with CTX
exposure was calreticulin. These proteins have been reported to participate in cell
defense and protection mechanisms. Proteomic analysis of toxic and relatively nontoxic
G. undulatus revealed that four proteins were down-regulated including
crocalbin-like protein, nephrocystin-6 and glutathione S-transferase (GST). The
altered proteins are responsible for detoxification, intracellular homeostastis, and
immune defense.
GL-a cells exposed to P-CTX-1 for 48 hours showed differences in their
protein expression profiles. Forty-two proteins were identified and matched with the
Epinephelus coioides protein sequence database. Eight proteins showed a doseresponse
relationship, of which, triosephosphate isomerase B, ribosomal protein LPlike
protein, AHNAK nucleoprotein isoform 1 and natural killer cell enhancement
factor showed dose-dependent suppression. In the in vivo exposure study of juvenile
groupers, ten fish proteins from the liver were found to be up-regulated after 3 ng/g
exposure, whereas three proteins were down-regulated. Five proteins were highly
expressed in fish from the 30 ng/g exposure, while four proteins were suppressed.
The altered proteins are responsible for detoxification, intracellular homeostastis,
and immune defense.
To conclude, this study reports the ciguatoxicity of coral reef fish collected
from the Republic of Kiribati as determined using the MNA. Nine-fold greater
ciguatoxicity was present in liver from highly toxic moray eels compared to muscle,
suggesting that this tissue is suitable for proteomic studies of CTX exposure. Based
on proteomic analysis, two groups of proteins were identified to show changes
associated with CTX-exposure and were commonly identified among samples,
suggesting they are potential markers for P-CTX-1 exposure. The first group is
composed of Ca2+ homeostasis and regulation proteins, including crocalbin-like
protein, calreticulin, AHNAK nucleoprotein, natural killer cell enhancement factor
and actin. Another group of proteins consists of the iron-binding proteins: ferritin
and transferrin. The present work demonstrates that proteomics provides a new
insight into the molecular events associated with CTX exposure in reef fish.
| Date of Award | 15 Feb 2012 |
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| Original language | English |
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| Awarding Institution | - City University of Hong Kong
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| Supervisor | Kwan Sing Paul LAM (Supervisor) |
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