Long-Term, Low-Dose Exposure to Microcystin-LR Does not Cause or Increase the Severity of Liver Disease in Rodents
Meaghan Labine*, Yuewen Gong**, Gerald Y. Minuk
,
Corresponding author
Gerald.Minuk@umanitoba.ca
Correspondence and reprint request: G.Y. Minuk, M.D. Morberg Family Chair in Hepatology University of Manitoba. John Buhler Research Centre 715 McDermot Ave. Winnipeg, MB R3E 3P4 Phone: (204) 789-3204. Fax: (204) 789-3987
Correspondence and reprint request: G.Y. Minuk, M.D. Morberg Family Chair in Hepatology University of Manitoba. John Buhler Research Centre 715 McDermot Ave. Winnipeg, MB R3E 3P4 Phone: (204) 789-3204. Fax: (204) 789-3987
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Minuk" "autores" => array:3 [ 0 => array:3 [ "nombre" => "Meaghan" "apellidos" => "Labine" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">*</span>" "identificador" => "aff1" ] ] ] 1 => array:3 [ "nombre" => "Yuewen" "apellidos" => "Gong" "referencia" => array:1 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">**</span>" "identificador" => "aff2" ] ] ] 2 => array:4 [ "nombre" => "Gerald Y." "apellidos" => "Minuk" "email" => array:1 [ 0 => "Gerald.Minuk@umanitoba.ca" ] "referencia" => array:2 [ 0 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">*</span>" "identificador" => "aff1" ] 1 => array:2 [ "etiqueta" => "<span class="elsevierStyleSup">*</span>" "identificador" => "cor1" ] ] ] ] "afiliaciones" => array:2 [ 0 => array:3 [ "entidad" => "Department of Pharmacology and Therapeutics, Faculties of Medicine. University of Manitoba,Winnipeg,Manitoba,Canada" "etiqueta" => "*" "identificador" => "aff1" ] 1 => array:3 [ "entidad" => "Pharmacy. University of Manitoba,Winnipeg,Manitoba,Canada" "etiqueta" => "**" "identificador" => "aff2" ] ] "correspondencia" => array:1 [ 0 => array:3 [ "identificador" => "cor1" "etiqueta" => "*" "correspondencia" => "Correspondence and reprint request: G.Y. Minuk, M.D. Morberg Family Chair in Hepatology University of Manitoba. John Buhler Research Centre 715 McDermot Ave. Winnipeg, MB R3E 3P4 Phone: (204) 789-3204. Fax: (204) 789-3987" ] ] ] ] "resumenGrafico" => array:2 [ "original" => 0 "multimedia" => array:7 [ "identificador" => "f0015" "etiqueta" => "Figure 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 658 "Ancho" => 965 "Tamanyo" => 58328 ] ] "descripcion" => array:1 [ "en" => "<p id="sp0015" class="elsevierStyleSimplePara elsevierViewall">Oxidative stress of CFSC-2G hepatic stellate cells following 24 hours exposure to varying concentrations of microcystin-LR.</p>" ] ] ] "textoCompleto" => "<span class="elsevierStyleSections"><span id="s0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0040">Introduction</span><p id="p0010" class="elsevierStylePara elsevierViewall">Cyanobacteria (blue-green algae) contamination of drinking water is an increasingly common public health concern throughout the world.<a class="elsevierStyleCrossRefs" href="#bib0010"><span class="elsevierStyleSup">1,2</span></a> Previous case reports and animal studies have documented that acute exposure to various cyanobacterial toxins (cyanotoxins) and microcystins (MC) in particular, can cause severe hepa-tocyte injury and liver failure.<a class="elsevierStyleCrossRefs" href="#bib0020"><span class="elsevierStyleSup">3-5</span></a> Based on these findings, the World Health Organization (WHO) designated 1.0 μg/L of MC-LR as the maximum safe concentration of cyanotoxin permissible in human drinking water.<a class="elsevierStyleCrossRef" href="#bib0035"><span class="elsevierStyleSup">6</span></a></p><p id="p0015" class="elsevierStylePara elsevierViewall">The mechanism whereby cyanotoxins induce hepa-tocyte injury is unclear but appears to involve increased oxidative stress, inhibition of serine/threonine phos-phatase activity and/or activation of Akt and p38/ERK/ JNK signaling.<a class="elsevierStyleCrossRefs" href="#bib0040"><span class="elsevierStyleSup">7-11</span></a> There are also data to suggest that sub-lethal exposures to cyanotoxins enhance hepatic lipotoxicity and are fibrogenic in the liver and possibly the heart by activating tissue myofibroblasts.<a class="elsevierStyleCrossRefs" href="#bib0060"><span class="elsevierStyleSup">11-15</span></a></p><p id="p0020" class="elsevierStylePara elsevierViewall">In the present study we documented the effects of long-term, low-dose exposure to MC-LR in mice with healthy livers and those with thioacetamide (TAA)-in-duced liver injury. We also documented the <span class="elsevierStyleItalic">in vitro</span> effects of a range of MC-LR concentrations on the metabolic activity, proliferation and activation of hepatic stellate cells (HSC), the myofibroblasts of the liver principally responsible for hepatic fibrogenesis.</p></span><span id="s0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0045">Material and Methods</span><p id="p0025" class="elsevierStylePara elsevierViewall">Adult male CD1 mice weighing 20-24 g, obtained from the University of Manitoba Animal Care Facility, were acclimatized for two weeks prior to use. Animals had free access to commercial laboratory chow and were maintained on a 12 h light:dark cycle throughout the study.</p><p id="p0030" class="elsevierStylePara elsevierViewall">Four study groups (N = 20/group) were analyzed: those allowed free access to water alone, water containing 1.0 μg/L of MC-LR (Sigma Aldrich, Oakville, ON, Canada), 1.0 μg/L of MC-LR and 300 mg/L of TAA (Sigma Aldrich) or 300 mg/L of TAA alone. Following 28 weeks of exposure, mice were euthanized, body/liver weights recorded, blood tested by standard biochemical techniques for evidence of hepatic inflammation (serum alanine aminotransferase - ALT) and dysfunction (serum total bilirubin - TB) and hepatic histology was examined. The latter consisted of staining 5 μm slices of paraffin embedded tissue with Hemotoxylin and Eosin (H&E) and Picric Acid Sirius Red for evidence of inflammatory activity and fibrosis respectively. H&E slides were graded for inflammation according to the metavir inflammation scoring scale using the following units; 0 = no inflammation, 1 = minimal inflammation/ occasional spotty necrosis, 2 = mild inflammation/little hepatocellular damage, 3 = moderate inflammation with noticeable hepatocellular damage and 4 = severe inflammation with prominent diffuse hepatocellular damage. Picric Acid and Sirius Red stained slides were graded for fibrosis according to the metavir fibrosis grade scale using the following units; 0 = no scarring, 1 = fibrosis confined to the portal tracts, 2 = fibrosis extending beyond the portal tracts, 3 = bridging fibrosis; fibrosis spreading and connecting to central veins or other portal tracts and 4 = cirrhosis or advanced scarring of the liver.</p><p id="p0035" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">In vitro</span> studies were performed with a rodent hepatic stellate cell (HSC) line (CFSC-2G), a gift from Dr. Y. Gong, Faculty of Pharmacy, University of Manitoba. Cells were cultured in Dulbecco's Modified Eagle's medium (DMEM) (Gibco Laboratories, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS), 100 units/mL of penicillin and 100 μg/mL streptomycin and incubated at 37 °C (in a humidifier in a 5% C0<span class="elsevierStyleInf">2</span> atmosphere) until tested for oxidative stress, ser-ine/threonine phosphatase activity, proliferation and activation (transition to a myofibroblast phenotype).</p><span id="s0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0050">Oxidative Stress</span><p id="p0040" class="elsevierStylePara elsevierViewall">CFSC-2G cells were plated in 96 well plates at a density of 10,000 cells per well in 100 μ/L of supplemented DMEM medium and allowed to adhere overnight. Medium was replaced with medium containing various concentrations of MC-LR (0.1-10,000 μg/L), 0.1 μg/L of okadaic acid (OA) (positive control) or medium alone. After 24 h of incubation at 37 °C, CellRox Deep Red reagent oxidative stress dye (Life Technologies, Grand Island, NY, USA) was added to each well at a concentration of 5 μM and incubated at 37 °C for 30 min according to manufacturer's instructions. Cells were then washed x3 using sterile PBS. Readings were taken using a Ziess Axio observer Z1 fluorescent microscope at a 40x objective against untreated cells containing PBS and 5 μM of CellRox dye and analyzed using Ziess AxioVision 4 software.</p></span><span id="s0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0055">Serine/Threonine Phosphatase Inhibition</span><p id="p0045" class="elsevierStylePara elsevierViewall">CFSC-2G cells were seeded at 200,000 cells/well in DMEM supplemented medium in 6 well plates and allowed to attach overnight. Medium containing various concentrations of MC-LR (0.1- 10,000 μg/L), okadaic acid (positive control) or medium alone was then added. Cells were allowed to incubate for an additional 24 hrs, washed with sterile PBS, and lysed using radioim-munoprecipitation (RIPA) lysis buffer plus protease inhibitor. Cell lysates were then added to a RediPlate 96 EnzChek Serine/threonine Phosphatase Assay Kit Plate (Molecular Probes, Eugene, OR, United States) as per the manufacturer's instructions. Fluorescence readings were measured using a BioTek Synergy 4 microplate reader (BioTek Instruments, Winoosky, VT, USA) at an excitation/emission maxima of 358/452.</p></span><span id="s0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0060">Cellular Proliferation</span><p id="p0050" class="elsevierStylePara elsevierViewall">WST-1[2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] (WST-1) (Roche Applied Science, Laval, QC, Canada) was employed to document CFSC-2G proliferative activity. Briefly, 96 well plates were seeded with CFSC-2G cells at a density of 2,000 cells/well in DMEM medium and allowed to attach for 4 h. Medium was then replaced with medium containing various concentrations of MC-LR (0.1-1,000 μg/L), Epidermal Growth Factor (100 μg/L) or Platelet Derived Growth Factor (100 μg/L) (positive controls). Following 24, 48, 72 and 96 h incubation at 37 °C, cells were washed with PBS and fresh medium containing 10 μ/L of the WST-1 reagent was added to each well. Cells were then incubated for 2 h at 37 °C, and absorbance read at 450 nm (reference wavelength 630 nm) using a BioTek plate reader (BioTek Instruments). Readings were compared against cell free blanks containing medium alone and WST-1 reagent.</p></span><span id="s0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0065">Activation</span><p id="p0055" class="elsevierStylePara elsevierViewall">Activation of HSCs to a myofibroblast phenotype was determined by documenting the expression of smooth muscle actin alpha (SMAA) protein. Following exposure to MC-LR or positive controls as described above for cell proliferation, CFSC-2G cells were lysed using RIPA lysis buffer plus protease inhibitor. Protein levels were quantified using a Bradford Protein Assay Kit as per the manufacturer's instructions (Bio-Rad Laboratories, Mississauga, ON, Canada). Readings were taken at an absorbance of 560 nm (reference wavelength optical density 630 nm) using a BioTek plate reader (BioTek Instruments). Aliquots were electrophoresed through 10% polyacrylamide-SDS gels and resolved proteins transferred to Nitro-pins 2,000 membranes (Micron Separations, Westborough, MA, USA). Membranes were blocked with 5.0% skim milk in tris-buff-ered saline for 1 h at room temperature, incubated overnight at 4 °C in the presence of 1:1000 anti-SMAA (Roche Molecular Biochemicals, Laval, QC, Canada), diluted in PBS + 1% Tween-20. Membranes were then washed in PBS + 1% Tween-20 5x, exposed to peroxi-dase-conjugated sheep anti-mouse secondary antibody (1:1000 diluted in PBS + 1% Tween-20) (Amersham-Pharmacia Biotech, Piscataway, NJ, USA) for 1 h at room temperature and washed in PBS +1% Tween 20 5x. Bands were detected using an enhanced chemilumi-nescence system (Amersham-Pharmacia Biotech, Pis-cataway, NJ, USA).</p></span></span><span id="s0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0070">Statistics</span><p id="p0060" class="elsevierStylePara elsevierViewall">Body/liver weights and plasma chemistry comparisons were performed with Sigma Plot software applying one-way anova analysis. Liver histology results for inflammation and fibrosis were analyzed for significance using the Kruskal-Wallis one-way anova on ranks method, comparing the treatment groups to negative controls. A two-sided P < 0.05 was considered significant.</p></span><span id="s0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0075">Results</span><p id="p0065" class="elsevierStylePara elsevierViewall"><a class="elsevierStyleCrossRef" href="#t0010">Table 1</a> provides the results of body and liver weight determinations at the time of sacrifice. Compared to water alone exposed mice, MC-LR exposed mice had similar body and liver weights. However, MC-LR/TAA and TAA alone exposed groups had significantly lower body weights and higher liver/body weight ratios when compared to water alone exposed controls (p < 0.01 respectively).</p><elsevierMultimedia ident="t0010"></elsevierMultimedia><p id="p0070" class="elsevierStylePara elsevierViewall">Serum ALT and TB determinations were also measured at the time of sacrifice. Levels were similar in water and MC-LR alone exposed mice but significantly elevated in the MC-LR/TAA and TAA alone exposed cohorts (p < 0.05). The extent of the elevations in ALT and TB values were similar in the latter two cohorts.</p><p id="p0075" class="elsevierStylePara elsevierViewall">The results of H&E staining for hepatic inflammation are provided in <a class="elsevierStyleCrossRef" href="#f0010">figure 1</a> and semi-quantitatively in <a class="elsevierStyleCrossRef" href="#t0015">table 2</a>. There was either no or minimal evidence of hepatic inflammatory activity in the livers of water and MC-LR alone exposed mice while inflammation was significantly increased (but to a similar extent) in the MC-LR/TAA and TAA alone cohorts (METAVIR scores: 2.6 ± 1.0 and 3.3 ± 0.7 respectively) when compared to water alone controls (p < 0.05 respectively).</p><elsevierMultimedia ident="f0010"></elsevierMultimedia><elsevierMultimedia ident="t0015"></elsevierMultimedia><p id="p0080" class="elsevierStylePara elsevierViewall">Picric Sirius Red staining for fibrosis (<a class="elsevierStyleCrossRef" href="#f0010">Figure 1</a> and <a class="elsevierStyleCrossRef" href="#t0015">Table 2</a>) indicated no differences in the extent of fibro-sis in livers of water and MC-LR alone exposed mice but significant increases (again, to a similar extent) in the MC-LR/TAA and TAA alone cohorts (METAVIR scores: 2.0 ± 1.0 and 1.8 ± 0.7 respectively) when compared to water alone controls (p < 0.01 respectively).</p><p id="p0085" class="elsevierStylePara elsevierViewall">The results of HSC exposure to a range of MC-LR concentrations <span class="elsevierStyleItalic">in vitro</span> are provided in figures 2-5. As shown in <a class="elsevierStyleCrossRefs" href="#f0015">figures 2 and 3</a>, only at the highest concentrations of MC-LR tested (5,000 and 10,000 μg/L) were significant increases in oxidative stress and inhibition of serine/threonine phosphatase activity observed. Regarding proliferative activity, there were no increases in HSC proliferation after 24, 48 and 96 h of exposure to MC-LR at concentrations as high as 1,000 μg/L compared to buffer alone controls (<a class="elsevierStyleCrossRef" href="#f0025">Figure 4</a>). Similarly, SMAA protein expression remained unaltered following exposure to the same concentrations of MC-LR for identical durations of time (<a class="elsevierStyleCrossRef" href="#f0030">Figure 5</a>).</p><elsevierMultimedia ident="f0015"></elsevierMultimedia><elsevierMultimedia ident="f0020"></elsevierMultimedia><elsevierMultimedia ident="f0025"></elsevierMultimedia><elsevierMultimedia ident="f0030"></elsevierMultimedia></span><span id="s0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0080">Discussion</span><p id="p0090" class="elsevierStylePara elsevierViewall">The results of this study indicate that healthy mice exposed to a low concentration of MC-LR in their drinking water for a total of 28 wks (approximately 30% of their anticipated life span) do not develop enlarged livers, biochemical evidence of active hepatic inflammation or dysfunction and have essentially normal liver histology. The results also suggest that long-term, low-dose exposure does not potentiate hepatic inflammation or fibrosis in the setting of active (TAA induced) liver disease. These findings were supported by the results of <span class="elsevierStyleItalic">in vitro</span> experiments which demonstrated that low concentrations of MC-LR do not induce oxidative stress, inhibit serine/threonine phosphatase activity, enhance the proliferative activity or activate HSCs to undergo transformation to a myofibroblast phenotype.</p><p id="p0095" class="elsevierStylePara elsevierViewall">Although the above results are reassuring, it must be noted they are not consistent with previous findings from other investigators. Specifically, Elleman, <span class="elsevierStyleItalic">et al</span>. described hepatocyte degeneration, scattered lobular necrosis, mononuclear cell infiltration and progressive fibrosis in mice treated daily for six weeks with “sub-lethal” intraperitoneal injections of purified MC-LR at 25%, 50% and 75% of the LD100 dose.<a class="elsevierStyleCrossRef" href="#bib0070"><span class="elsevierStyleSup">13</span></a> In another study, Frangez, <span class="elsevierStyleItalic">et al</span>. reported increased peri-portal inflammation and fibrosis in female New Zealand rabbits treated every other day for three weeks with intraperi-toneal injections of 7.5 mg/kg cyanobacterial lysates containing 1 mg/g of MC-RR.<a class="elsevierStyleCrossRef" href="#bib0075"><span class="elsevierStyleSup">14</span></a> Finally, He, <span class="elsevierStyleItalic">et al</span>. described changes in keeping with nonalcoholic steato-hepatitis in BALB/c mice exposed to low-dose (40 μg/ kg) MD-LR for 90 days.<a class="elsevierStyleCrossRef" href="#bib0060"><span class="elsevierStyleSup">11</span></a></p><p id="p0100" class="elsevierStylePara elsevierViewall">The reason(s) for the discrepancy between the above reports and our own findings remain to be determined. Whether differences in species (mice versus rabbits), strains of mice (CD1 versus BALB/c), nutrition (in the above studies MC-exposed mice lost significant amounts of weight) and routes of administration (oral <span class="elsevierStyleItalic">versus</span> intraperitoneal) explain the different outcomes requires further research. Also to be considered are differences in the concentrations of MC-LR employed. Unfortunately, the previous studies provided insufficient data to calculate the molarity of MC-LR required for comparative analyses. Finally, it is possible that exposure to more than one MC congener is required to cause inflammation and/or fibrosis, as suggested by Frangez, <span class="elsevierStyleItalic">et al</span>.<a class="elsevierStyleCrossRef" href="#bib0080"><span class="elsevierStyleSup">15</span></a></p><p id="p0105" class="elsevierStylePara elsevierViewall">The need to determine whether cyanotoxins enhance hepatic injury in the setting of existing liver disease was driven by the growing epidemic of obesity and high prevalence of viral hepatitis which together, have resulted in large segments of the general population being diagnosed with chronic liver disease. Thus, whether long-term, low-dose cyanotoxin exposure enhances hepatic inflammation and fibrosis in the setting of existing liver disease is an important clinical question that had yet to be addressed. Here again, the results of the present study are reassuring. Biochemical and histolog-ic evidence of hepatic inflammation and fibrosis were similar in MC-LR/TAA compared to TAA alone exposed mice. Whether the same findings would be obtained in other models of chronic liver disease remains to be determined.</p><p id="p0110" class="elsevierStylePara elsevierViewall">There are a number of limitations to this study that warrant emphasis. First, only the most common and well-studied cyanotoxin, MC-LR, was employed. Second, the amount of water spillage in the animal holding cages was not measured and therefore, precise determinations of MC-LR exposure could not be made. Third, the in vitro studies involved HSCs alone and perhaps co-cultures with hepatocytes or other non-parenchymal cells would have provided different results. Finally, properly designed studies in humans where cyanotoxin contamination of the drinking water has been documented (and quantitated) are required to address the question whether these encouraging findings in rodents can be extrapolated to humans.</p><p id="p0115" class="elsevierStylePara elsevierViewall">In conclusion, the results of the present study do not support concerns that long-term, low-dose exposure to cyanotoxins cause hepatic inflammation or fibrosis in healthy livers or exacerbate either feature in the setting of existing liver disease.</p></span><span id="s0055" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0085">Acknowledgements</span><p id="p0120" class="elsevierStylePara elsevierViewall">This research was supported by a grant from the Public Health Agency of Canada. Ms M. Labine is a recipient of a Manitoba Health Research Council fellowship award. The authors would also like to thank Ms R. Vizniak for her prompt and accurate typing of the manuscript.</p></span><span id="s0060" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0090">Compliance with Ethical Standards</span><p id="p0125" class="elsevierStylePara elsevierViewall">There was no conflict of interest. Approval for the study was obtained from the University of Manitoba animal ethics committee. Funding was received from a graduate studentship from Manitoba Water Stewardship for Meaghan Labine.</p></span></span>" "textoCompletoSecciones" => array:1 [ "secciones" => array:10 [ 0 => array:3 [ "identificador" => "xres1199350" "titulo" => "Abstract" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "abs0010" "titulo" => "Background" ] 1 => array:2 [ "identificador" => "abs0015" "titulo" => "Material and methods" ] 2 => array:2 [ "identificador" => "abs0020" "titulo" => "Results" ] 3 => array:2 [ "identificador" => "abs0025" "titulo" => "Conclusion" ] ] ] 1 => array:2 [ "identificador" => "xpalclavsec1117807" "titulo" => "Keywords" ] 2 => array:2 [ "identificador" => "s0010" "titulo" => "Introduction" ] 3 => array:3 [ "identificador" => "s0015" "titulo" => "Material and Methods" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "s0020" "titulo" => "Oxidative Stress" ] 1 => array:2 [ "identificador" => "s0025" "titulo" => "Serine/Threonine Phosphatase Inhibition" ] 2 => array:2 [ "identificador" => "s0030" "titulo" => "Cellular Proliferation" ] 3 => array:2 [ "identificador" => "s0035" "titulo" => "Activation" ] ] ] 4 => array:2 [ "identificador" => "s0040" "titulo" => "Statistics" ] 5 => array:2 [ "identificador" => "s0045" "titulo" => "Results" ] 6 => array:2 [ "identificador" => "s0050" "titulo" => "Discussion" ] 7 => array:2 [ "identificador" => "s0055" "titulo" => "Acknowledgements" ] 8 => array:2 [ "identificador" => "s0060" "titulo" => "Compliance with Ethical Standards" ] 9 => array:1 [ "titulo" => "References" ] ] ] "pdfFichero" => "main.pdf" "tienePdf" => true "fechaRecibido" => "2016-12-15" "fechaAceptado" => "2017-04-16" "PalabrasClave" => array:1 [ "en" => array:1 [ 0 => array:4 [ "clase" => "keyword" "titulo" => "Keywords" "identificador" => "xpalclavsec1117807" "palabras" => array:8 [ 0 => "Cyanobacteria" 1 => "Blue-green algae" 2 => "Cyanotoxins" 3 => "Microcystins" 4 => "Hepatic fibrosis" 5 => "Cirrhosis" 6 => "Chronic liver disease" 7 => "Hepatic stellate cells" ] ] ] ] "tieneResumen" => true "resumen" => array:1 [ "en" => array:3 [ "titulo" => "Abstract" "resumen" => "<span id="abs0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0015">Background</span><p id="sp0045" class="elsevierStyleSimplePara elsevierViewall">Acute exposure to high concentrations of microcystin-LR (MC-LR) can cause significant hepatocyte injury. Aim. To document the effects of long-term, low-dose MC-LR exposure on hepatic inflammation and fibrosis in mice with healthy and diseased livers.</p></span> <span id="abs0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0020">Material and methods</span><p id="sp0050" class="elsevierStyleSimplePara elsevierViewall">Male CD1 mice (N = 20/group) were exposed to 1.0 μg/L of MC-LR in drinking water; 1.0 μg/L MC-LR plus 300 mg/L of the hepatotoxin thioacetamide (MC-LR/TAA); or 300 mg/L TAA alone for 28 weeks. Liver biochemistry and histology were documented at the end of the study period. In addition, hepatic stellate cells (HSCs), were exposed <span class="elsevierStyleItalic">in vitro</span> to MC-LR (0.1-10,000 μg/L) and monitored for changes in cell metabolism, proliferation and activation.</p></span> <span id="abs0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0025">Results</span><p id="sp0055" class="elsevierStyleSimplePara elsevierViewall">Liver biochemistry and histology were essentially normal in MC-LR alone exposed mice. MC-LR/TAA and TAA alone exposed mice had significant hepatic inflammation and fibrosis but the extent of the changes were similar in the two groups. <span class="elsevierStyleItalic">In vitro</span>, MC-LR had no effect on HSC metabolism, proliferation or activation.</p></span> <span id="abs0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="st0030">Conclusion</span><p id="sp0060" class="elsevierStyleSimplePara elsevierViewall">Long-term, low-dose exposure to MC-LR is unlikely to lead to chronic liver disease in the setting of a normal liver or exacerbate existing liver disease in the setting of ongoing hepatitis.</p></span>" "secciones" => array:4 [ 0 => array:2 [ "identificador" => "abs0010" "titulo" => "Background" ] 1 => array:2 [ "identificador" => "abs0015" "titulo" => "Material and methods" ] 2 => array:2 [ "identificador" => "abs0020" "titulo" => "Results" ] 3 => array:2 [ "identificador" => "abs0025" "titulo" => "Conclusion" ] ] ] ] "multimedia" => array:7 [ 0 => array:7 [ "identificador" => "f0010" "etiqueta" => "Figure 1" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr1.jpeg" "Alto" => 792 "Ancho" => 2074 "Tamanyo" => 220874 ] ] "descripcion" => array:1 [ "en" => "<p id="sp0010" class="elsevierStyleSimplePara elsevierViewall">Hematoxylin & Eosin and Picric Cirius stains of livers of male CD1 mice exposed to water alone (control) microcystin-LR (MC-LR) alone, MC-LR plus thioacetamide (TAA) or TAA alone for 28 weeks.</p>" ] ] 1 => array:7 [ "identificador" => "f0015" "etiqueta" => "Figure 2" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr2.jpeg" "Alto" => 658 "Ancho" => 965 "Tamanyo" => 58328 ] ] "descripcion" => array:1 [ "en" => "<p id="sp0015" class="elsevierStyleSimplePara elsevierViewall">Oxidative stress of CFSC-2G hepatic stellate cells following 24 hours exposure to varying concentrations of microcystin-LR.</p>" ] ] 2 => array:7 [ "identificador" => "f0020" "etiqueta" => "Figure 3" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr3.jpeg" "Alto" => 655 "Ancho" => 977 "Tamanyo" => 80438 ] ] "descripcion" => array:1 [ "en" => "<p id="sp0020" class="elsevierStyleSimplePara elsevierViewall">Serine/threonine phosphatase 2A (PP2A) activity in CFSC-2G hepatic stellate cells following 24 hours exposure to varying concentrations of microcystin-LR.</p>" ] ] 3 => array:7 [ "identificador" => "f0025" "etiqueta" => "Figure 4" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr4.jpeg" "Alto" => 613 "Ancho" => 2072 "Tamanyo" => 113655 ] ] "descripcion" => array:1 [ "en" => "<p id="sp0025" class="elsevierStyleSimplePara elsevierViewall">Proliferative activity of CFSC-2G hepatic stellate cells following 96 h exposure to varying concentrations of microcystin-LR.</p>" ] ] 4 => array:7 [ "identificador" => "f0030" "etiqueta" => "Figure 5" "tipo" => "MULTIMEDIAFIGURA" "mostrarFloat" => true "mostrarDisplay" => false "figura" => array:1 [ 0 => array:4 [ "imagen" => "gr5.jpeg" "Alto" => 1382 "Ancho" => 1713 "Tamanyo" => 143363 ] ] "descripcion" => array:1 [ "en" => "<p id="sp0030" class="elsevierStyleSimplePara elsevierViewall">Smooth muscle alpha actin protein (SMAA) expression in CFSC-2G hepatic stellate cells following 96 h exposure to varying concentrations of microcystin-LR.</p>" ] ] 5 => array:7 [ "identificador" => "t0010" "etiqueta" => "Table 1" "tipo" => "MULTIMEDIATABLA" "mostrarFloat" => true "mostrarDisplay" => false "tabla" => array:2 [ "tablatextoimagen" => array:1 [ 0 => array:2 [ "tabla" => array:1 [ 0 => """ <table border="0" frame="\n \t\t\t\t\tvoid\n \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><th class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Treament group \t\t\t\t\t\t\n \t\t\t\t\t\t</th><th class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" scope="col" style="border-bottom: 2px solid black">BWT \t\t\t\t\t\t\n \t\t\t\t\t\t</th><th class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" scope="col" style="border-bottom: 2px solid black">LWT \t\t\t\t\t\t\n \t\t\t\t\t\t</th><th class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" scope="col" style="border-bottom: 2px solid black">LWT % BWT \t\t\t\t\t\t\n \t\t\t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">Control \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">53.8 ± 5.3 g \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">2.8 ± 0.3 g \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">5.0 ± 0.1% \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">MC-LR \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">55.8 ± 5.2 g \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">2.7 ± 0.6 g \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">4.8 ± 0.1% \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">MC-LR + TAA \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">45.7 ± 4.1 g<a class="elsevierStyleCrossRef" href="#tbl1fn1"><span class="elsevierStyleSup">*</span></a> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">3.2 ± 0.6 g \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">6.0 ± 0.1%<a class="elsevierStyleCrossRef" href="#tbl1fn2"><span class="elsevierStyleSup">**</span></a> \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">TAA \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">47.4 ± 3.5 g<a class="elsevierStyleCrossRef" href="#tbl1fn1"><span class="elsevierStyleSup">*</span></a> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">3.1 ± 0.6 g \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">6.5 ± 0.2%<a class="elsevierStyleCrossRef" href="#tbl1fn2"><span class="elsevierStyleSup">**</span></a> \t\t\t\t\t\t\n \t\t\t\t</td></tr></tbody></table> """ ] "imagenFichero" => array:1 [ 0 => "xTab2048477.png" ] ] ] "notaPie" => array:2 [ 0 => array:3 [ "identificador" => "tbl1fn1" "etiqueta" => "*" "nota" => "<p class="elsevierStyleNotepara" id="np0010">P < 0.05</p>" ] 1 => array:3 [ "identificador" => "tbl1fn2" "etiqueta" => "**" "nota" => "<p class="elsevierStyleNotepara" id="np0015">P < 0.01, BWT: Body weight. LWT: Liver weight. MC-LR: Microcystin-LR. TAA: Thioa cetamide.</p>" ] ] ] "descripcion" => array:1 [ "en" => "<p id="sp0035" class="elsevierStyleSimplePara elsevierViewall">Body Weight, Liver Weight and Percent Liver to Body Wight in Mice Exposed to Water Alone (control), MC-LR Alone, MC-LR plus TAA or TAA Alone for 28 Weeks.</p>" ] ] 6 => array:7 [ "identificador" => "t0015" "etiqueta" => "Table 2" "tipo" => "MULTIMEDIATABLA" "mostrarFloat" => true "mostrarDisplay" => false "tabla" => array:2 [ "tablatextoimagen" => array:1 [ 0 => array:2 [ "tabla" => array:1 [ 0 => """ <table border="0" frame="\n \t\t\t\t\tvoid\n \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><th class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Treatment group \t\t\t\t\t\t\n \t\t\t\t\t\t</th><th class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" scope="col" style="border-bottom: 2px solid black">ALT (< 50) \t\t\t\t\t\t\n \t\t\t\t\t\t</th><th class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Total bilirubin (0.4) \t\t\t\t\t\t\n \t\t\t\t\t\t</th><th class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Inflammation grade (0.4) \t\t\t\t\t\t\n \t\t\t\t\t\t</th><th class="td" title="\n \t\t\t\t\ttable-head\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Fibrosis grade (0.4) \t\t\t\t\t\t\n \t\t\t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">Control \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">43.61 ± 17.48 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0.11 ± 0.03 mg/dL \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0.88 ± 0.89 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0.06 ± 0.25 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">MC-LR \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">48.35 ± 25.32 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0.12 ± 0.05 mg/dL \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">1.16 ± 0.5 \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0.26 ± 0.45 \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">MC-LR + TAA \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">69.12 ± 23.16<a class="elsevierStyleCrossRef" href="#tbl2fn1"><span class="elsevierStyleSup">*</span></a> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0.22 ± 0.04 mg/dL<a class="elsevierStyleCrossRef" href="#tbl2fn2"><span class="elsevierStyleSup">**</span></a> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">2.57 ± 0.99<a class="elsevierStyleCrossRef" href="#tbl2fn2"><span class="elsevierStyleSup">**</span></a> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">2 ± 1.03<a class="elsevierStyleCrossRef" href="#tbl2fn2"><span class="elsevierStyleSup">**</span></a> \t\t\t\t\t\t\n \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">TAA \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">68.71 ± 25.71<a class="elsevierStyleCrossRef" href="#tbl2fn1"><span class="elsevierStyleSup">*</span></a> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">0.21 ± 0.03 mg/dL<a class="elsevierStyleCrossRef" href="#tbl2fn2"><span class="elsevierStyleSup">**</span></a> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">3.26 ± 0.65<a class="elsevierStyleCrossRef" href="#tbl2fn2"><span class="elsevierStyleSup">**</span></a> \t\t\t\t\t\t\n \t\t\t\t</td><td class="td" title="\n \t\t\t\t\ttable-entry\n \t\t\t\t " align="left" valign="\n \t\t\t\t\ttop\n \t\t\t\t">1.79 ± 0.71<a class="elsevierStyleCrossRef" href="#tbl2fn2"><span class="elsevierStyleSup">**</span></a> \t\t\t\t\t\t\n \t\t\t\t</td></tr></tbody></table> """ ] "imagenFichero" => array:1 [ 0 => "xTab2048478.png" ] ] ] "notaPie" => array:2 [ 0 => array:3 [ "identificador" => "tbl2fn1" "etiqueta" => "*" "nota" => "<p class="elsevierStyleNotepara" id="np0020">P < 0.05</p>" ] 1 => array:3 [ "identificador" => "tbl2fn2" "etiqueta" => "**" "nota" => "<p class="elsevierStyleNotepara" id="np0025">P < 0.01, ALT: Alanine aminotransferase. MC-LR: Microcystin-LR. TAA: Thiocentamide.</p>" ] ] ] "descripcion" => array:1 [ "en" => "<p id="sp0040" class="elsevierStyleSimplePara elsevierViewall">Serum Alanine Aminotransferase, Total Bilirubin, Histologic Grade and Stage of Liver Disease in Mice Exposed to Water Alone (control), MC-LR Alone, MC-LR + TAA or TAA Alopne for 28 weeks.</p>" ] ] ] "bibliografia" => array:2 [ "titulo" => "References" "seccion" => array:1 [ 0 => array:2 [ "identificador" => "bs0010" "bibliografiaReferencia" => array:15 [ 0 => array:3 [ "identificador" => "bib0010" "etiqueta" => "1" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "Toxicology and risk assessment of freshwater cyanobacterial (blue-green algal) toxins in water" "autores" => array:1 [ 0 => array:2 [ "etal" => false "autores" => array:4 [ 0 => "Duy TN" 1 => "Lam PK" 2 => "Shaw GR" 3 => "Connell DW" ] ] ] ] ] "host" => array:1 [ 0 => array:1 [ "Revista" => array:6 [ "tituloSerie" => "Rev Environ Contam Toxicol" "fecha" => "2000" "volumen" => "163" "paginaInicial" => "113" "paginaFinal" => "185" "link" => array:1 [ 0 => array:2 [ "url" => "https://www.ncbi.nlm.nih.gov/pubmed/10771585" "web" => "Medline" ] ] ] ] ] ] ] ] 1 => array:3 [ "identificador" => "bib0015" "etiqueta" => "2" "referencia" => array:1 [ 0 => array:2 [ "contribucion" => array:1 [ 0 => array:2 [ "titulo" => "Health Effects of Toxin-Producing Cy-anobacteria: The CyanoHABs. 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| Year/Month | Html | Total | |
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| 2024 April | 22 | 11 | 33 |
| 2024 March | 21 | 3 | 24 |
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| 2024 January | 8 | 5 | 13 |
| 2023 December | 8 | 6 | 14 |
| 2023 November | 12 | 6 | 18 |
| 2023 October | 17 | 6 | 23 |
| 2023 September | 8 | 1 | 9 |
| 2023 August | 6 | 5 | 11 |
| 2023 July | 8 | 7 | 15 |
| 2023 June | 23 | 5 | 28 |
| 2023 May | 38 | 7 | 45 |
| 2023 April | 51 | 3 | 54 |
| 2023 March | 39 | 5 | 44 |
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| 2022 March | 32 | 19 | 51 |
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| 2020 May | 25 | 7 | 32 |
| 2020 April | 21 | 2 | 23 |
| 2020 March | 23 | 0 | 23 |
| 2020 February | 21 | 4 | 25 |
| 2020 January | 19 | 2 | 21 |
| 2019 December | 10 | 8 | 18 |
| 2019 November | 3 | 1 | 4 |
| 2019 October | 9 | 1 | 10 |
| 2019 September | 12 | 1 | 13 |
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