Effects of MP Polyethylene Microparticles on Microbiome and Inflammatory Response of Larval Zebrafish.

Plastic polymers have quickly become one of the most abundant materials on Earth due to their low production cost and high versatility. Unfortunately, some of the discarded plastic can make its way into the environment and become fragmented into smaller microscopic particles, termed secondary microplastics (MP). In addition, primary MP, purposely manufactured microscopic plastic particles, can also make their way into our environment via various routes. Owing to their size and resilience, these MP can then be easily ingested by living organisms. The effect of MP particles on living organisms is suspected to have negative implications, especially during early development. In this study, we examined the effects of polyethylene MP ingestion for four and ten days of exposure starting at 5 days post-fertilization (dpf). In particular, we examined the effects of polyethylene MP exposure on resting metabolic rate, on gene expression of several inflammatory and oxidative stress linked genes, and on microbiome composition between treatments. Overall, we found no evidence of broad metabolic disturbances or inflammatory markers in MP-exposed fish for either period of time. However, there was a significant increase in the oxidative stress mediator L-FABP that occurred at 15 dpf. Furthermore, the microbiome was disrupted by MP exposure, with evidence of an increased abundance of Bacteroidetes in MP fish, a combination frequently found in intestinal pathologies. Thus, it appears that acute polyethylene MP exposure can increase oxidative stress and dysbiosis, which may render the animal more susceptible to diseases.

Molecular cloning and functional characterization of a glucose transporter (CsGLUT) in Clonorchis sinensis.

A complementary DNA (cDNA) encoding a glucose transporter of Clonorchis sinensis (CsGLUT) was isolated from the adult C. sinensis cDNA library. The open reading frame of CsGLUT cDNA consists of 1653 base pairs that encode a 550-amino acid residue protein. Hydropathy analysis suggested that CsGLUT possess 12 putative membrane-spanning domains. The Northern blot analysis result using poly(A)(+)RNA showed a strong band at ~2.1 kb for CsGLUT. When expressed in Xenopus oocytes, CsGLUT mediated the transport of radiolabeled deoxy-D-glucose in a time-dependent but sodium-independent manner. Concentration-dependency results showed saturable kinetics and followed the Michaelis-Menten equation. Nonlinear regression analyses yielded a Km value of 588.5 ± 53.0 µM and a Vmax value of 1500.0 ± 67.5 pmol/oocyte/30 min for [1,2-(3)H]2-deoxy-D-glucose. No trans-uptakes of bile acid (taurocholic acid), amino acids (tryptophan and arginine), or p-aminohippuric acid were observed. CsGLUT-mediated transport of deoxyglucose was significantly and concentration-dependently inhibited by radio-unlabeled deoxyglucose and D-glucose. 3-O-Methylglucose at 10 and 100 µM inhibited deoxyglucose uptake by ~50 % without concentration dependence. No inhibitory effects by galactose, mannose, and fructose were observed. This work may contribute to the molecular biological study of carbohydrate metabolism and new drug development of C. sinensis.

Resistance to the macrocyclic lactone moxidectin is mediated in part by membrane transporter P-glycoproteins: Implications for control of drug resistant parasitic nematodes.

Our objective was to determine if the resistance mechanism to moxidectin (MOX) is similar of that to ivermectin (IVM) and involves P-glycoproteins (PGPs). Several Caenorhabditis elegans strains were used: an IVM and MOX sensitive strain, 13 PGP deletion strains and the IVM-R strain which shows synthetic resistance to IVM (by creation of three point mutations in genes coding for α-subunits of glutamate gated chloride channels [GluCls]) and cross-resistance to MOX. These strains were used to compare expression of PGP genes, measure motility and pharyngeal pumping phenotypes and evaluate the ability of compounds that inhibit PGP function to potentiate sensitivity or reverse resistance to MOX. The results suggest that C. elegans may use regulation of PGPs as a response mechanism to MOX. This was indicated by the over-expression of several PGPs in both drug sensitive and IVM-R strains and the significant changes in phenotype in the IVM-R strain in the presence of PGP inhibitors. However, as the inhibitors did not completely disrupt expression of the phenotypic traits in the IVM-R strain, this suggests that there likely are multiple avenues for MOX action that may include receptors other than GluCls. If MOX resistance was mediated solely by GluCls then exposure of the IVM-R strain to PGP inhibitors should not have affected sensitivity to MOX. Targeted gene deletions showed that protection of C. elegans against MOX involves complex mechanisms and depends on the PGP gene family, particularly PGP-6. While the results presented are similar to others using IVM, there were some important differences observed with respect to PGPs which may play a role in the disparities seen in the characteristics of resistance to IVM and MOX. The similarities are of concern as parasites resistant to IVM show some degree but not complete cross-resistance to MOX; this could impact nematodes that are resistant to IVM.

Immunity against selected piscine flagellates.

This discussion is on immune response to Amyloodinium ocellatum, Cryptobia salmositica, Trypanoplasma borreli and Trypanosoma carassii. Piscidin and histone-like proteins enhance innate resistance to Amyloodinium. Fish that are naturally resistant to Cryptobia and Trypanoplasma can be bred. Cryptobia resistance in charr is controlled by a dominant Mendelian locus and protection is via the Alternative Pathway of Complement Activation. Studies on Cryptobia-tolerant charr may lead to production of transgenic Cryptobia-tolerant salmon. Innate response to T. borreli is associated with NO in macrophages. Transferrin regulates resistance and carp have been bred for transferrin genotypes. Recovered fish are protected from homologous challenge, and complement fixing antibodies are crucial in protection. Studies on antigens in T. carassii may lead to a vaccine. There are two vaccines against cryptobiosis; a single dose of the attenuated vaccine protects salmonids. On challenge fish inoculated with the metalloprotease-DNA vaccine do not have the disease and they recover faster.

Transport proteins of the ABC systems superfamily and their role in drug action and resistance in nematodes.

The completion of a number of nematode genomes has provided significant information on ABC systems in these organisms. Nematodes have more ABC systems genes and greater diversity than do mammalian species. Class 1 and class 2 ABC systems, more commonly known as ABC transporters, are present. As in other organisms, nematode ABC systems are characterized by a highly conserved ATP-binding domain (ABC_2) and a less conserved transmembrane domain (ABC_TM1/TM1F). Studies of drug resistance in nematodes have suggested that ABC transporters are part of the resistance mechanism. Evidence in support of this has been obtained from genetic studies where an association between anthelmintic selection and ABC transporters was shown by comparisons between unselected and drug selected, or resistant, populations of parasitic nematodes. In drug resistant populations, genetic polymorphism and diversity, genotype patterns, and linkage disequilibrium were disrupted. Multidrug resistance (MDR) reversing agents that inhibit ABC function improve efficacy in sensitive nematode populations and restore sensitivity in resistant populations. Similar to the situation in clinical oncology, overexpression of ABC systems occurs in drug resistant and sensitive populations following drug exposure, particularly those in the P-glycoprotein (PGP) subfamily. Deletion or disruption of ABC genes, particularly PGP and the multidrug resistance associated protein (MRP), increases sensitivity to some drugs, particularly ivermectin. These studies provide evidence that ABC transporters play a role in drug action and resistance in nematodes.

Inhibition of P-glycoprotein enhances sensitivity of Caenorhabditis elegans to ivermectin.

In vertebrates, the function of P-glycoprotein (PGP) is to protect against toxic compounds through active efflux of the toxin from target tissues. In clinical oncology, the overexpression of PGP confers drug resistance. The function(s) of PGP in nematode physiology or in conferring drug resistance is less understood. The objective of this study was to determine the role of PGP in drug resistance in nematodes using Caenorhabditis elegans and ivermectin (IVM) as the model system. The IVM sensitive wild-type Bristol N2 strain, seven PGP deletion strains and a triple IVM receptor (avr-14/avr-15/glc-1) knock-out strain showing synthetic resistance to IVM (IVM-R) were used to (1) compare the gene expression signatures of 15 PGPs in the wild-type and resistant strains following treatment; (2) measure motility and pharyngeal pumping phenotypes in the wild-type, IVM-R and PGP deletion strains before and after treatment; and (3) quantify the phenotypic responses of the wild-type and IVM-R strains to IVM or IVM co-administered with 12 chemosensitizers that interfere with PGP function. IVM induced changes in both amplitude and timing of gene expression for the 15 PGP genes. Following IVM treatment, the most significant effects were observed in the IVM-R strain for those PGP genes expressed in the neurons, pharynx and intestine. Inactivation of pgp-2, pgp-5, pgp-6, pgp-7, pgp-12 and pgp-13 resulted in increased sensitivity to IVM compared with the wild-type. The phenotypic responses of the IVM-R strain differed from those of the wild-type strain when exposed to IVM alone, or IVM co-administered with chemosensitizers. The phenotypic responses to the co-administration of chemosensitizers varied with the concentration of IVM used, suggesting that the action of PGP's is influenced by the concentration of IVM. Verapamil restored sensitivity to IVM in the IVM-R strain. Our results demonstrate that PGPs play a role in protecting C. elegans from IVM toxicity and inhibition of PGP enhances susceptibility to IVM. PGP may be a mechanism for multidrug resistance (MDR) in parasitic nematodes.

The role of Brugia malayi ATP-binding cassette (ABC) transporters in potentiating drug sensitivity.

ATP binding cassette (ABC) systems are a diverse group of proteins that have been identified in every organism, from bacteria to humans. Analysis of nematode genomes indicates that the number and arrangement of ABC systems are similar to other organisms, with the majority being ABC transporters. There are few functional studies of ABC transporters in parasitic nematodes; most reports have been on their identification or use as genetic markers to monitor drug resistance. In eukaryotes, some ABC transporters function in tissue defense by actively removing drugs, thus preventing their accumulation. The overexpression of ABC transporters that function as efflux pumps, such as P-glycoprotein (PGP) and the multidrug resistance associated protein (MRP) are known to confer resistance. Drug sensitivity can be restored by administration of PGP interfering or MDR reversal agents. The objective of this study was to determine if ABC systems in filarioid nematodes function similarly to those of other organisms. The relative expression of 33 ABC systems identified in Brugia malayi was quantified following exogenous exposure to the commonly used drug ivermectin (IVM). Following exposure of adults and microfilariae to IVM, there was a significant increase in the transcriptional profiles of a number of ABC systems, mostly within the PGP and MRP subgroups. Coadministration of PGP-interfering and MDR-reversal agents with IVM potentiated sensitivity to the drug in adults and microfilariae. The results suggest that B. malayi ABC transporters function similarly to those in other organisms and are a factor in determining drug sensitivity.

A comparison of the effects of ivermectin and moxidectin on the nematode Caenorhabditis elegans.

The avermectins and the milbemycins are structurally related classes of 16-membered macrocyclic lactones (ML) that have a broad spectrum of activity. Most studies on the mode of action of ML have used the avermectin, ivermectin (IVM). IVM activates glutamate-gated chloride channels that contain alpha-type subunits, resulting in a hyperpolarization of the neuronal membrane, leading to a flaccid paralysis. IVM kills Caenorhabditis elegans at therapeutic concentrations, making it a useful model to examine mechanisms of IVM toxicity and resistance. There have been suggestions that the milbemycins may exert effects that are different from the avermectins, however this hypothesis has been challenged. Using IVM and the milbemycin, moxidectin (MOX), we demonstrate that while the two drugs have some similar effects on C. elegans, there are also some differences in worm response. Following exogenous exposure to a gradient of IVM and MOX, ranging from 0 to 5000 nM, quantitative and qualitative differences in response to the two anthelmintic drugs were observed in the pharyngeal pump rate, larval development and motility of wild-type and glutamate-gated chloride channel (GluCl) subunit knockout strains of C. elegans. After exposure to equimolar drug concentrations, differences between the anthelmintic effects were observed in the motility phenotype in the wild-type, GluCl subunit knockout strains and multi-gene knockout strain of C. elegans that exhibits a marked reduction in IVM sensitivity; and transcription profiles of genes coding for GluCl subunits in both the wild-type and glc-2 knockout strain. The glc-2 deletion strain showed increased motility in response to 2.5nM MOX in the first 1.5h of exposure, compared with wild-type nematodes, whereas this strain showed little change in motility in response to IVM. The pharyngeal pump rate in the glc-2 deletion strain was sensitive to equimolar concentrations of IVM and MOX. The triple avr-14/avr-15/glc-1 knockout caused a loss of initial stimulation of motility seen in the wild-type, by 2.5 nM IVM, to a reduction in motility, whereas the response to MOX was little changed between this triple knockout strain and wild-type C. elegans. The results suggest that there are significant differences in the response of C. elegans to IVM and MOX. The product of the glc-2 gene may play a role in sensitivity to MOX, but not to IVM, while the products of avr-14, avr-15 and glc-1 may be important for the effects of IVM, but less so for MOX.

P-glycoprotein-like protein, a possible genetic marker for ivermectin resistance selection in Onchocerca volvulus.

Ivermectin (IVM) is the only safe drug for mass-treatment of onchocerciasis. IVM resistance has been reported in gastrointestinal nematode parasites of animals. A reduction in response to IVM in Onchocerca volvulus could have significant consequences for the onchocerciasis control programs. We have found evidence that, in O. volvulus, repeated IVM treatment selects for specific alleles, of P-glycoprotein-like protein (PLP), a half-sized ABC transporter. In this study, O. volvulus samples were derived from a clinical trial in Cameroon, in which patients were sampled before, and following 3 years (1994-1997) of IVM treatments. There were four treatment groups: 150 microg/kg (1 x p.a. or 4 x p.a.) and 800 microg/kg (1 x p.a. or 4 x p.a.). DNA of O. volvulus macrofilariae was genotyped over a 476bp region of the PLP gene and at two control genes. Of the six polymorphic positions found in the PLP amplicon, three of them showed significant selection after 4 x p.a. treatment with IVM (total of 13 IVM treatments) in female worms, and one of the same single nucleotide polymorphisms (SNPs) showed significant selection in the male worms. One of the selected SNPs in the female worms caused an amino acid coding change in the putative protein sequence. We found a clear selection of some genotypes, a high SNPs association and a loss of polymorphism following 4 x p.a. treatment with IVM. These PLP SNPs and genotypes could be useful markers to follow selection for IVM resistance in the field.

Characterization of a half-size ATP-binding cassette transporter gene which may be a useful marker for ivermectin selection in Onchocerca volvulus.

ATP-binding cassette (ABC) transporters comprise a large paralogous protein family and several confer drug resistance. Ivermectin (IVM) is the only drug approved for treatment of onchocerciasis and is a substrate for some ABC transporters. Furthermore, there is accumulating evidence that IVM selects on some ABC transporter genes in Onchocerca volvulus and other parasitic nematodes. The onchocerciasis control programs rely on community based treatment with IVM each year to reduce morbidity and decrease parasite transmission. This appears to be imposing selection pressure on O. volvulus. A half-size ABC transporter cDNA has previously been reported for O. volvulus, however, the full length gene has not been previously characterized and investigated for possible selection by IVM. Genes under selection may be identified by patterns of linkage disequilibrium (LD) and a loss of genetic polymorphism at physically linked loci. Twelve genetic markers spanning the full gene were examined in O. volvulus from non-treated and IVM treated populations in Ghana. Analysis of the genomic organization of the half-size ABC transporter (OvPLP) indicates that it spans approximately 3.8 kb comprising nine exons. Worms from treated people showed a reduction in gene diversity, a loss of genetic polymorphism at several markers, a selection for specific alleles, and a reduction in the number of regions in LD; these effects were more pronounced as treatment increased. These changes suggest that IVM is imposing selection pressure on this gene. The region between transmembrane domains four and five may be a useful marker for IVM selection in O. volvulus.

Genomic organization and effects of ivermectin selection on Onchocerca volvulus P-glycoprotein.

Ivermectin (IVM) was first developed for use with livestock. It is now the only drug used for mass treatment of onchocerciasis. It is difficult to prove whether reports of sub-optimal responses to IVM in some Onchocerca volvulus infected patients are a result of drug resistance, as procedures typically used to examine IVM efficacy in livestock can not be performed on humans. To determine the effects of IVM on O. volvulus, one approach is to examine allele frequencies before and after treatment. Allele(s) linked to resistance may increase in frequency after repeated treatment. Mass treatment of large human populations to reduce transmission of O. volvulus will impose selection pressure for resistance. P-glycoprotein has been implicated as a candidate IVM resistance gene in nematodes. In this study, the intron-exon structure of O. volvulus P-glycoprotein (OvPGP) has been defined. The gene spans 10.6 kb, is AT-rich, contains 24 exons and a high proportion of class 0 introns. The genetic diversity of 28 loci spanning the entire OvPGP gene was examined in four O. volvulus populations from the Volta Region of Ghana. Worms collected in 1999 and 2002 from IVM treated patients showed reduced genetic polymorphism and an increase in the number of loci not in Hardy-Weinberg equilibrium. Changes in allelic patterns and a reduction in diversity at many loci in P-glycoprotein in the parasites from IVM treated patients in 1999 and 2002 suggest that IVM is imposing selection on this gene, consistent with a possible development of IVM resistance.