Proteomics: Applications and Advances 18.7 Algal Toxins 489
Part C | 18.7
2D-PAGE protein profiles were obtained for all species
and could be used to distinguish between closely related
species belonging to the same family, confirming 2DPAGE as an excellent monitoring tool for distinguishing
dinoflagellate species comprising a HAB [18.107].
Since some dinoflagellate species are morphologically similar, and/or toxic and nontoxic varieties of
the same species exist and sometimes even co-occur,
the ability to use 2D-PAGE to differentiate between
toxic and nontoxic variants of a species was investigated [18.106, 108]. Although the protein profiles of
a toxic and nontoxic strain of Alexandrium minutum
were essentially similar, reflecting the absence of significant morphological variation between the strains,
pronounced differences were detected [18.106]. Thus,
four proteins (designated NT1, NT2, NT3, and NT4)
could be consistently detected in all nontoxic strains,
while proteins T1 and T2 were associated with the toxic
strains regardless of the culture conditions.
A similar study, employing a combination of 2PAGE and MS was conducted on several strains
of Alexandrium tamarense with different toxin compositions and originating from different geographical locations, identified several proteins that were
always present in all toxic Alexandrium tamarense
strains [18.108]. Interestingly, MS analysis indicated
that the proteins associated with toxic Alexandrium
tamarense are isoforms of the same protein, which
shares amino acid similarity to that of T1 from toxic
Alexandriunm minutum. Since the peptide mass fingerprints of these toxicity biomarkers were highly strainand species-specific, differentiating between toxic and
nontoxic strains as well as Alexandrium tamarense and
Alexandrium minutum, the study has made a significant contribution to the complex task of monitoring
HABs for toxic species. Indeed, the use of these toxicity biomarkers was validated by western blot experiments using a murine AT-T1-polycolonal antibody
that detected all the toxic Alexandrium tamarense and
Alexandrium minutum strains, but not the nontoxic
Alexandrium tamarense strain [18.108].
Besides monitoring HABs in the ocean, it is also
of utmost importance to monitor seafood destined for
human consumption for toxin contamination. Although
this may be accomplished using analytical techniques,
the diversity of toxins and their analogs that may contaminate seafood makes this approach problematic due
to the lack of specific standards and reference compounds required to ensure the accuracy and specificity
of the protocol. Consequently, Ronzitti et al. [18.109]
investigated a proteomics approach that would provide a functional assay for toxin-contaminated seafood
by screening for toxin biomarkers in situ. They used
2D-PAGE to analyze digestive gland samples obtained
from mussels harvested from four sites in the Adriatic Sea for biomarkers of contamination with okadaic
acid and related dinophysis toxins, responsible for diarrhoeic shellfish poisoning in humans. Two proteins
that correlated with okadaic acid contamination of the
digestive gland samples were identified as a component of photosystem II and a subunit of NADH
dehydrogenase, the former being identical to its counterpart in Dinophysis species that produce this group of
toxins [18.109].
Ronzitti et al. [18.109] concluded that the photosystem II component originated from Dinophysis dinoflagellates ingested by the filter feeding mussels, and the
amount ingested is reflected by the quantity of this protein which correlated with the degree of okadaic acid
contamination of the mussel digestive gland samples
(as determined by chemical analysis). Thus the study
demonstrated the feasibility of biomarkers for monitoring seafood, where the biomarkers can either reflect the
origin of the toxin (photosystem II component) or the
animal’s response to the toxin (NADH dehydrogenase
subunit – possibly functions as a component of a protective response to the presence of a strong apoptogenic
compound in the mussel digestive gland) [18.109].
Besides inducing diarrhoeic shellfish poisoning in
humans after consuming bivalves contaminated with
Dinophysis dinoflagellates, okadaic acid has been recognized as a general tumor promoter in various murine
organs, including the skin, glandular stomach, and
liver [18.110, 111]. Although the genes and signaling pathways affected by okadaic acid toxicity have
been elucidated using a mouse toxicological microarray that identified a total of 177 differentially expressed
genes [18.111], almost all the available knowledge concerning the toxicological mechanisms of okadaic acid
has been obtained from in vitro assays. Furthermore,
because the effects of okadaic acid on cell transformation, cell proliferation, and apoptosis vary considerably,
the molecular events underlying the effects of okadaic
acid are not well understood [18.110, 111]. Consequently, Wang et al. [18.110] employed 2D-DIGE and
MALDI-TOF-TOF MS to investigate the acute toxicity
of okadaic acid in mice at the proteomic level to further
elucidate the molecular mechanism of okadaic acidinduced diarrhoea in mammals. Male Imprinting Control Region (ICR) mice were treated with 750 g=kg
okadaic acid, administered in a single dose by gavage,
and protein profiles of the small intestines were de-
Part C | 18.7
2D-PAGE protein profiles were obtained for all species
and could be used to distinguish between closely related
species belonging to the same family, confirming 2DPAGE as an excellent monitoring tool for distinguishing
dinoflagellate species comprising a HAB [18.107].
Since some dinoflagellate species are morphologically similar, and/or toxic and nontoxic varieties of
the same species exist and sometimes even co-occur,
the ability to use 2D-PAGE to differentiate between
toxic and nontoxic variants of a species was investigated [18.106, 108]. Although the protein profiles of
a toxic and nontoxic strain of Alexandrium minutum
were essentially similar, reflecting the absence of significant morphological variation between the strains,
pronounced differences were detected [18.106]. Thus,
four proteins (designated NT1, NT2, NT3, and NT4)
could be consistently detected in all nontoxic strains,
while proteins T1 and T2 were associated with the toxic
strains regardless of the culture conditions.
A similar study, employing a combination of 2PAGE and MS was conducted on several strains
of Alexandrium tamarense with different toxin compositions and originating from different geographical locations, identified several proteins that were
always present in all toxic Alexandrium tamarense
strains [18.108]. Interestingly, MS analysis indicated
that the proteins associated with toxic Alexandrium
tamarense are isoforms of the same protein, which
shares amino acid similarity to that of T1 from toxic
Alexandriunm minutum. Since the peptide mass fingerprints of these toxicity biomarkers were highly strainand species-specific, differentiating between toxic and
nontoxic strains as well as Alexandrium tamarense and
Alexandrium minutum, the study has made a significant contribution to the complex task of monitoring
HABs for toxic species. Indeed, the use of these toxicity biomarkers was validated by western blot experiments using a murine AT-T1-polycolonal antibody
that detected all the toxic Alexandrium tamarense and
Alexandrium minutum strains, but not the nontoxic
Alexandrium tamarense strain [18.108].
Besides monitoring HABs in the ocean, it is also
of utmost importance to monitor seafood destined for
human consumption for toxin contamination. Although
this may be accomplished using analytical techniques,
the diversity of toxins and their analogs that may contaminate seafood makes this approach problematic due
to the lack of specific standards and reference compounds required to ensure the accuracy and specificity
of the protocol. Consequently, Ronzitti et al. [18.109]
investigated a proteomics approach that would provide a functional assay for toxin-contaminated seafood
by screening for toxin biomarkers in situ. They used
2D-PAGE to analyze digestive gland samples obtained
from mussels harvested from four sites in the Adriatic Sea for biomarkers of contamination with okadaic
acid and related dinophysis toxins, responsible for diarrhoeic shellfish poisoning in humans. Two proteins
that correlated with okadaic acid contamination of the
digestive gland samples were identified as a component of photosystem II and a subunit of NADH
dehydrogenase, the former being identical to its counterpart in Dinophysis species that produce this group of
toxins [18.109].
Ronzitti et al. [18.109] concluded that the photosystem II component originated from Dinophysis dinoflagellates ingested by the filter feeding mussels, and the
amount ingested is reflected by the quantity of this protein which correlated with the degree of okadaic acid
contamination of the mussel digestive gland samples
(as determined by chemical analysis). Thus the study
demonstrated the feasibility of biomarkers for monitoring seafood, where the biomarkers can either reflect the
origin of the toxin (photosystem II component) or the
animal’s response to the toxin (NADH dehydrogenase
subunit – possibly functions as a component of a protective response to the presence of a strong apoptogenic
compound in the mussel digestive gland) [18.109].
Besides inducing diarrhoeic shellfish poisoning in
humans after consuming bivalves contaminated with
Dinophysis dinoflagellates, okadaic acid has been recognized as a general tumor promoter in various murine
organs, including the skin, glandular stomach, and
liver [18.110, 111]. Although the genes and signaling pathways affected by okadaic acid toxicity have
been elucidated using a mouse toxicological microarray that identified a total of 177 differentially expressed
genes [18.111], almost all the available knowledge concerning the toxicological mechanisms of okadaic acid
has been obtained from in vitro assays. Furthermore,
because the effects of okadaic acid on cell transformation, cell proliferation, and apoptosis vary considerably,
the molecular events underlying the effects of okadaic
acid are not well understood [18.110, 111]. Consequently, Wang et al. [18.110] employed 2D-DIGE and
MALDI-TOF-TOF MS to investigate the acute toxicity
of okadaic acid in mice at the proteomic level to further
elucidate the molecular mechanism of okadaic acidinduced diarrhoea in mammals. Male Imprinting Control Region (ICR) mice were treated with 750 g=kg
okadaic acid, administered in a single dose by gavage,
and protein profiles of the small intestines were de-