The effects of single l-amino acid substitutions on the lethal potencies of the microcystins

doi:10.1016/0041-0101(89)90051-2

Toxicon

Volume 27, Issue 7, 1989, Pages 825-828

https://doi.org/10.1016/0041-0101(89)90051-2 Get rights and content

Abstract

R. D. Stoner, W. H. Adams, D. N. Slatkin and H. W. Siegelman. The effects of single l-amino acid substitutions on the lethal potencies of the microcystins. Toxicon27, 825–828, 1989.—Microcystin-LR and -LA are more toxic than microcystin-LY and -RR in adult mice. They induce different degrees of thrombocytopenia and leukopenia, and the lethalities of their binary and ternary mixtures are additive. Postnatal mice are resistant to doses of microcystin-LR that are lethal to adults but they are susceptible to higher doses. Substitution of a single l-amino acid for another in a microcystin markedly affects the dosimetric potency, but not the pathophysiology of its toxicity.

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Cited by (55)

Occurrence and toxicity of microcystin congeners other than MC-LR and MC-RR: A review
2019, Food and Chemical Toxicology
Citation Excerpt :
These differences in toxicity have also been observed in vivo. Stoner et al. (1989) found that MC-LR and MC-LA were more toxic than MC-LY and MC-RR in adult mice. Substitution of a single l-amino acid for another markedly affected the dosimetric potency, but not the pathophysiology of its toxicity.
The occurrence of cyanobacterial toxins is being increasingly reported. This is a reason for concern as they can induce toxic effects both in humans and in the environment. Among them, microcystins (MCs) are the best described and most diverse group of cyanobacterial toxins, and MC-LR and MC-RR are the congeners most widely investigated. However, the number of MC variants has also increased in recent years. Some of these minority variants have been shown to have a different toxicokinetic and toxicodynamic profile, but research focused on them is still limited. Moreover, in some water bodies these minority variants can be the predominant toxins. Nonetheless, MC-LR is the only one used for risk evaluation purposes at present. In order to contribute to more realistic risk assessments in the future, the aim of this review was to compile the available information in the scientific literature regarding the occurrence and concentration of minority MCs in water and food samples, and their toxic effects. The data retrieved demonstrate the congener-specific toxicity of MCs, as well as many data gaps in relation to analytical or mechanistic aspects, among others. Therefore, further research is needed to improve the toxicological characterization of these toxins and the exposure scenarios.
Analysis of free and metabolized microcystins in samples following a bird mortality event
2018, Harmful Algae
Citation Excerpt :
Experimental toxicity data on birds is limited, and factors such as route of exposure, dose and intra/inter-species variability requires consideration when reviewing the literature. Takahashi and Kaya (1993) reported the LD50 (i.p.) of MC-RR in quail to be 256 μg kg−1 (b.w.), similar to the 111–650 μg kg−1 (b.w.) range for MC-RR in mice (Chen et al., 2006; Gupta et al., 2003; Stoner et al., 1989; Stotts et al., 1993; Watanabe et al., 1988). In other studies, dosing of quail and ducks was conducted using crude extracts or cyanobacterial biomass, with only acute sub-lethal responses observed, including liver damage, oxidative stress and enlarged spleens (Damkova et al., 2009; Kral et al., 2012; Li et al., 2012; Pašková et al., 2008; Skocovska et al., 2007).
In the summer of 2012, over 750 dead and dying birds were observed at the Paul S. Sarbanes Ecosystem Restoration Project at Poplar Island, Maryland, USA (Chesapeake Bay). Clinical signs suggested avian botulism, but an ongoing dense Microcystis bloom was present in an impoundment on the island. Enzyme-linked immunosorbent assay (ELISA) analysis of a water sample indicated 6000 ng mL⁻¹ of microcystins (MCs). LC-UV/MS analysis confirmed the presence of MC-LR and a high concentration of an unknown MC congener (m/z 1037.5). The unknown MC was purified and confirmed to be [D-Leu¹]MC-LR using NMR spectroscopy, LC-HRMS and LC–MS², which slowly converted to [D-Leu¹,Glu(OMe)⁶]MC-LR during storage in MeOH. Lyophilized algal material from the bloom was further characterized using LC-HRMS and LC–MS² in combination with chemical derivatizations, and an additional 24 variants were detected, including MCs conjugated to Cys, GSH and γ-GluCys and their corresponding sulfoxides. Mallard (Anas platyrhynchos) livers were tested to confirm MC exposure. Two broad-specificity MC ELISAs and LC–MS² were used to measure free MCs, while ‘total’ MCs were estimated by both MMPB (3-methoxy-2-methyl-4-phenylbutyric acid) and thiol de-conjugation techniques. Free microcystins in the livers (63–112 ng g⁻¹) accounted for 33–41% of total microcystins detected by de-conjugation and MMPB techniques. Free [D-Leu¹]MC-LR was quantitated in tissues at 25–67 ng g⁻¹ (LC–MS²). The levels of microcystin varied based on analytical method used, highlighting the need to develop a comprehensive analysis strategy to elucidate the etiology of bird mortality events when microcystin-producing HABs are present.
Transgenerational effects of cyanobacterial toxins on a tropical micro-crustacean Daphnia lumholtzi across three generations
2018, Environmental Pollution
Citation Excerpt :
Again, we could not observe this in the treatment of the F0 for which we cannot provide a plausible explanation at this point. Though MCs are very potently toxic to aquatic animals (Stoner et al., 1989; Oberemm et al., 1999) other cyanobacterial metabolites from extract might have generated the observed effects, but we didn't have the possibility to determine in the current study. Continuous exposure to both MC-LR and cyanobacterial extract resulted in aggravated effects on fitness-related traits of F1 generation.
Climate change and human activities induce an increased frequency and intensity of cyanobacterial blooms which could release toxins to aquatic ecosystems. Zooplankton communities belong to the first affected organisms, but in tropical freshwater ecosystems, this issue has yet been poorly investigated. We tested two questions (i) if the tropical Daphnia lumholtzi is capable to develop tolerance to an ecologically relevant concentration of purified microcystin-LR and microcystins from cyanobacterial extract transferable to F1 and F2 generations? And (ii) would F1 and F2 generations recover if reared in toxin-free medium? To answer these questions, we conducted two full factorial mutigenerational experiments, in which D. lumholtzi was exposed to MC-LR and cyanobacterial extract at the concentration of 1 μg L⁻¹ microcystin continuously for three generations. After each generation, each treatment was spit into two: one reared in the control (toxin free) while the other continued in the respective exposure. Fitness-related traits including survival, maturity age, body length, and fecundity of each D. lumholtzi generation were quantified. Though there were only some weak negative effects of the toxins on the first generation (F0), we found strong direct, accumulated and carried-over impacts of the toxins on life history traits of D. lumholtzi on the F1 and F2, including reductions of survival, and reproduction. The maturity age and body length showed some inconsistent patterns between generations and need further investigations. The survival, maturity age (for extract), and body length (for MC-LR) were only recovered when offspring from toxin exposed mothers were raised in clean medium for two generations. Chronic exposure to long lasting blooms, even at low density, evidently reduces survival of D. lumholtzi in tropical lakes and reservoirs with ecological consequences.
Improved screening of microcystin genes and toxins in blue-green algal dietary supplements with PCR and a surface plasmon resonance biosensor
2015, Harmful Algae
Citation Excerpt :
While these studies establish SPR as a potential screening method, the current immunoassays are limited by (1) long assay times (60 min cycle (Herranz et al., 2010), approx. 17 min cycle for a single measurement (Vinogradova et al., 2011)), (2) only testing water samples and not complex dietary supplements (Herranz et al., 2010), and/or (3) use of an R-specific antibody (Herranz et al., 2010; Vinogradova et al., 2011). Recent studies by FDA researchers and others (Heussner et al., 2012) have indicated that MC-LA and MC-LF may also be present in BGA supplements, and MC-LA has been reported to be equally potent to MC-LR (Stoner et al., 1989). Therefore, an assay using the R-specific Ab would miss these congeners that do not contain an arginine group and potentially underreport sample toxicity.
Microcystins (MCs) comprise a group of cyclic heptapeptide toxins that share a common backbone and have two variable l-amino acids that yield at least 21 known analogs of varying potency. These hepatotoxins and potential tumor promoters are produced by certain cyanobacteria, including Microcystis aeruginosa. The cyanobacterium M. aeruginosa blooms in freshwater lakes and can potentially co-occur with other species such as Aphanizomenon flos-aquae, which is targeted and harvested for the production of dietary supplements known as blue-green algae (BGA). BGA supplements are currently marketed in the U.S. and internationally as a product that may elevate mood, increase energy, and alleviate attention deficit hyperactivity disorder. However, the potential for BGA dietary supplements to be contaminated with MCs is of concern, and there are currently no validated methods for detection of MCs in these products. This research focused on establishing screening methods for toxic Microcystis and MCs in BGA supplements. A DNA-based method employing polymerase chain reaction (PCR) was used as a prescreening tool to evaluate the dietary supplements and to detect the presence of toxin genes (i.e., presence of toxic Microcystis). A rapid, sensitive surface plasmon resonance (SPR) biosensor, directed towards recognition of all MC forms, was also developed and validated. This improved SPR biosensor incorporates a commercial Adda-group antibody (Ab) that has the capacity for broader recognition of MCs than previously developed sensors for BGA supplements that rely solely on an arginine-reactive Ab and can quantitate MC levels down to 0.24 ng/mL (equivalent to 0.24 μg per gram of BGA supplement) in less than 10 min. Such a rapid, quantitative screening method may allow for further surveillance of BGA products to assist risk assessment efforts, establishment of regulatory guidance levels, and response to potential consumer complaints related to BGA products. The PCR technique and SPR biosensor may be used in concert as prescreening and screening tools, respectively or individually, thereby limiting the number of samples that must be evaluated with confirmatory methods.
Identification of microcystins from three collection strains of Microcystis aeruginosa
2010, Environmental Pollution
Citation Excerpt :
Even though the 7 possible amino acids corresponding to m/z ion 1009.6 were identified, more assays are necessary to confirm the MC-HilR assignation. The peak at m/z 1002.5 (MC-3, Table 3) could correspond to three known MCs: MC-LY (Stoner et al., 1989), [L-Ser7] MC-EE(OMe) (Namikoshi et al., 1998) and [D-Asp3, Ser7] MC-E(OMe)E(OMe) (Namikoshi et al., 1998). In this case, the observed Tyr-like UV spectrum (maximum absorption at 232 nm, not shown), suggested the MC-LY ascription.
Microcystins (MCs) are toxic cyclic heptapeptides produced by various cyanobacteria genera, especially Microcystis. We identified 10 out of 12 MCs produced by three Microcystis aeruginosa strains from cyanobacteria collections, UTEX 2666, UTEX 2670 and UAM 1303, by using two analytical methods: Matrix-assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF/MS) and HPLC Photodiode Array Detector coupled to a hybrid Quadrupole Time of Flight Mass Spectrometry (HPLC-PDA-QTOF/MS). MALDI-TOF/MS failed to detect non-polar MCs, such as MC-LY and MC-LW. HPLC-QTOF/MS permitted the accurate identification of most MCs present in methanolic extracts. Besides, three new MCs, namely: [D-Glu(OCH₃)⁶, D-Asp³] MC-LAba, MC-YL and MC-YM were detected by HPLC-QTOF/MS.
The role of organic anion transporting polypeptides (OATPs/SLCOs) in the toxicity of different microcystin congeners in vitro: A comparison of primary human hepatocytes and OATP-transfected HEK293 cells
2010, Toxicology and Applied Pharmacology
Cellular uptake of microcystins (MCs), a family of cyclic cyanobacterial heptapeptide toxins, occurs via specific organic anion transporting polypeptides (OATPs), where MCs inhibit serine/threonine-specific protein phosphatase (PP). Despite comparable PP-inhibitory capacity, MCs differ greatly in their acute toxicity, thus raising the question whether this discrepancy results from MC-specific toxikokinetic rather than toxicodynamic differences. OATP-mediated uptake of MC congeners MCLR, -RR, -LW and –LF was compared in primary human hepatocytes and HEK293 cells stably expressing recombinant human OATP1B1/SLCO1B1 and OATP1B3/SLCO1B3 in the presence/absence of OATP substrates taurocholate (TC) and bromosulfophthalein (BSP) and measuring PP-inhibition and cytotoxicity. Control vector expressing HEK293 were resistant to MC cytotoxicity, while TC and BSP competition experiments reduced MC cytotoxicity in HEK293-OATP transfectants, thus confirming the requirement of OATPs for trans-membrane transport. Despite comparable PP-inhibiting capabilities, MCLW and -LF elicited cytotoxic effects at lower equimolar concentrations than MCLR and MCRR, hence suggesting congener selective transport into HEK293-OATP transfectants and primary human hepatocytes. Primary human hepatocytes appeared one order of magnitude more sensitive to MC congeners than the corresponding HEK293 -OATP transfectants. Although the latter maybe due to a much lower level of PPs in primary human hepatocytes, the presence of OATPs other than 1B1 or 1B3 may have added to an increased uptake of MCs. In view of the high sensitivity of human hepatocytes and currently MCLR-only based risk calculations, the actual risk of human MC-intoxication and ensuing liver damage could be underestimated in freshwater cyanobacterial blooms where MCLW and-LF predominate.

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Toxicon

Abstract

References (11)

Pathophysiological effects of a toxic peptide from Microcystis aeruginosa

Toxicon

Pathophysiology of cyanoginosin-LR: in vivo and in vitro studies

Toxicol. appl. Pharmac.

Monoclonal antibody specific for cyanoginosin-LA: preparation and characterization

Toxicon

Isolation and identification of the fast-death factor in Microcystis aeruginosa

Can. J. Biochem. Physiol.

Structural studies on cyanoginosins-LR, -YR, -YA, and -YM, peptide toxins from Microcystis aeruginosa

J. Chem. Soc. Perkin Trans.

Cited by (55)

Occurrence and toxicity of microcystin congeners other than MC-LR and MC-RR: A review

Analysis of free and metabolized microcystins in samples following a bird mortality event

Transgenerational effects of cyanobacterial toxins on a tropical micro-crustacean Daphnia lumholtzi across three generations

Improved screening of microcystin genes and toxins in blue-green algal dietary supplements with PCR and a surface plasmon resonance biosensor

Identification of microcystins from three collection strains of Microcystis aeruginosa

The role of organic anion transporting polypeptides (OATPs/SLCOs) in the toxicity of different microcystin congeners in vitro: A comparison of primary human hepatocytes and OATP-transfected HEK293 cells

Short communicationThe effects of single l-amino acid substitutions on the lethal potencies of the microcystins

Abstract

Toxicon

Toxicol. appl. Pharmac.

Toxicon

Isolation and identification of the fast-death factor in Microcystis aeruginosa

Can. J. Biochem. Physiol.

Structural studies on cyanoginosins-LR, -YR, -YA, and -YM, peptide toxins from Microcystis aeruginosa

J. Chem. Soc. Perkin Trans.

Short communication
The effects of single l-amino acid substitutions on the lethal potencies of the microcystins