* Department of Pharmacology, Environmental and Community Health Research, Research Institute of Pharmaceutical Sciences, University of Mississippi, University, Mississippi 38677;
Plymouth Marine Laboratory, Citadel Hill, Plymouth PL1 2PB, United Kingdom; and
USDA/ARS, Stuttgart National Aquaculture Center, Stuttgart, Arkansas 71260
Received August 4, 1999; accepted November 5, 1999
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ABSTRACT |
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Key Words: potassium permanganate; metallothionein; catfish; RT-PCR; 3`RACE; bankit 219393..
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INTRODUCTION |
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Metallothioneins (MTs) are low molecular weight proteins affording protection from metal toxicity and oxidative stress in numerous organisms (Kagi and Schaffer 1988). Although possessing a strong degree of structural homology throughout phylogeny, there appears to be significant differences in the expression of MT between species, especially in fish species (Olsson 1993
, 1996
). Recent studies exploring mechanisms of MT regulation have indicated differences in response elements that are important in initiating transcription of MT genes (Kille et al., 1991
). Channel catfish (Ictalurus punctatus) are fairly resistant to numerous chemicals including many organic pesticides and metals (Perkins and Schlenk, in press; Zhang and Schlenk, 1995). Earlier studies indicated relatively large intracellular concentrations of glutathione and antioxidant enzymes (Hasspieler et al., 1994
). However, little is known regarding the role of MT in the resistance of channel catfish to oxidatively active chemicals.
Previous studies showed that expression of hepatic MT is induced by several metals in channel catfish including cadmium (Zhang and Schlenk 1995), copper (Perkins et al., 1996), zinc (Schlenk et al.,1997a
), and arsenic (Schlenk et al.,1997b
). Although Mn has been shown to induce hepatic MT in vivo in rodents (Waalkes and Klaassen 1985
), it apparently does so through an indirect mechanism since levels of MT in isolated hepatocytes were actually reduced by Mn treatment without any change in cellular viability (Bracken and Klaassen 1987
).
MT expression can be measured at the level of the protein (Schlenk et al., 1997b) as well as mRNA (Schlenk et al., 1997a
; Zhang and Schlenk, 1995
). Recently, quantiative reverse-transcriptase polymerase chain reaction (RT-PCR) was used to measure expression of MT mRNA in channel catfish exposed to several metals (Schlenk et al., 1997a
). Unfortunately, the exact nucleotide sequence of the amplicon was never identified in any of these studies. Thus, the purposes of this study were to clone and sequence a previously identified cDNA obtained by RT-PCR which was induced by several metals, and to determine the relationship between expression of the transcript and sublethal whole animal endpoints indicative of toxicity in channel catfish.
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MATERIALS AND METHODS |
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Fish were offered a commercial catfish feed daily at the rate of 2% of body weight and uneaten feed was removed within 1 h. The manganese content of the feed used was 231 ± 18 mg/kg (n = 7 samples tested). Technical grade potassium permanganate used for this study was provided by Carus Chemical Company (Ottawa, IL); purity was rated at 97%.
Fish were transferred from holding tanks to 735-L cylindrical fiberglass tanks (124 x 61cm) and allowed to acclimate to the tanks and the environment for 2 weeks before manganese exposure. Flow rate of water into the experimental tanks was maintained at 12 tank volume turnovers per day (6.2 L/min). Three groups of 100 channel catfish (50 of each sex) weighing 400600 g each were exposed to waterborne potassium permanganate. One group was exposed to 0.5 mg potassium permanganate/L continuously during the study (8 weeks). Preliminary studies had shown that this concentration was the maximum that the fish could tolerate on a continuous basis. A second group was exposed to 1.0 mg potassium permanganate/L for 24 h every other day during the study and a third group was exposed to 2.0 mg potassium permanganate/L for 24 h every other day, via peristaltic pumps. A control group of 100 channel catfish was handled in the same manner as treated groups but the fish were not exposed to potassium permanganate. Manganese concentrations in tank waters were determined daily. Test samples were taken in triplicate at the upper, middle, and lower depths of tanks. Each sample was filtered through a new 0.10 µm pore nylon filter (Cameo 25NS, Micron Separations, Inc.) to remove manganese bound to particulates in the water. After filtration, each sample was acidified to 1% with nitric acid and analyzed by graphite furnace atomic absorption spectrophotometry (Thermo Jarrell Ash 1993). The quality control standard of Spex-QC-19 (Spex Industries, Inc., Edison, New Jersey) was used to establish standard curves for manganese concentrations in water.
Six catfish (3 males and 3 females) were removed from each group for sampling before initiating manganese exposure, and at 2-week intervals until the end of the study. Gender was determined by visual inspection of external genitalia. Fish were stunned by a blow to the head and the spinal cord was severed at the first cervical vertebra. Gills were collected from each fish, individually packaged in manganese-free sample holders, indexed, and stored at 85°C until analyzed for MT mRNA. The measurements of body weight, body length, liver weight, condition factor ((body weight (bw) x 100/bw3), and LSI (liver weight/[bw liver weight]) were recorded as measures of sublethal toxicity.
For RT-PCR studies, juvenile channel catfish (6 months old) of weights ranging from 100155 g were injected intraperitoneal with 10 mg/kg of cadmium chloride as previously described (Schlenk et al., 1997a). Following 24 h animals were euthanized with subsequent dissection of liver. Each tissue was frozen in liquid nitrogen and stored at 80°C until processed for RNA.
RNA Isolation and Northern Blotting
Total RNA was extracted from approximately 100 mg of fish gill using Tri reagent (Molecular Research Center, Inc., Cincinnnati, OH). The final RNA pellet was dissolved in DEPC-treated water and the concentration was measured using a Hitachi U-2000 spectrophotometer at 260 nm. The integrity of the RNA was checked by agarose gel electrophoresis by observation of 28S, 18S, and 5.8S ribosomal and transfer RNA bands. Northern blotting was performed using standard procedures previously described (Schlenk et al., 1997a,b
; Zhang and Schlenk, 1995
). Bands were quantitated by scanning densitometry using NIH Image software (version 1.61).
RT-PCR
Synthesis of the cDNA was carried out using the 1st Strand cDNA Synthesis kit (Boehringer Manheim, Indianopolis, IN) as previously described (Schlenk et al., 1997a). The RACE-T primer (5`-CCGAA TTCTC GAGAT CGATT TTTTT TTTTT TT-3`) was used to prime first strand cDNA synthesis. For PCR amplification, the RACE primer (5`CCGAA TTCTC GAGAT CGA-3`) and a degenerate, universal MT primer (5`-ATGGA TCCNT GCGAA TG-3`) based on the 6 N-terminal codons of piscine MT genes (Chan 1994
) were used. Primers were obtained from National Biosciences, Inc. (Plymouth MN). For the RT reaction, 1.0 µg of RNA was used in a total reaction volume of 50 µl containing 1x reaction buffer, 5 mM MgCl2, 1 mM DNTP mix, 50 Units of RNase inhibitor, 20 Units of AMV reverse transcriptase, and 10 ng of RACE-T primer. The reaction mixture was incubated at 42°C for 1 h and then diluted with 150 µl of water, to a total volume of 200 µl.
The PCR master mix contained 2.5 Units of Taq DNA polymerase in 20 mM TrisHCl, 100 mM KCl, 3 mM MgCl2, 0.01% Brijr 35 (v/v), dNTP mix (dATP, dCTP, dGTP, dTTP) each 0.4 mM, pH 8.3 (20°C) in a final volume of 50 µl. A set of specific primers for metallothionein expression was used as mentioned above. The PCR was performed using 25 cycles comprised of 3 segments of 94°C for 1 min; 50°C for 2 min; and 72°C for 3 min, and then a final extension at 70°C for 5 min. Temperature settings, cycle number, and RNA content were calibrated and optimized as previously described (Schlenk et al., 1997a).
Cloning and Sequencing
RT-PCR products were isolated from agarose gels using Millipore DNA extraction kits (Millipore, Bedford, MA) and cloned into the pCR-2.1 vector of the TA cloning System (Invitrogen, Carlsbad, CA). Plasmid DNA was prepared from 10 resulting bacterial colonies with a Qiagen Plasmid Mini-prep kit (Qiagen, Valencia, CA). The presence of appropriate sized inserts was confirmed by restriction enzyme digests with EcoRI (Promega, Madison WI). Six independently isolated clones were sequenced using a Themo Sequenase fluorescent labeled primer cycle sequencing kit (Amersham Pharmacia Biotech, Inc., Piscataway NJ) and IRD 700 labeled T7 or IRD 800 labeled M13-reverse sequencing primers (LI-COR, Inc., Lincoln NE). Sequencing reactions were run on a LI-COR 4200-L2 Sequencer (LI-COR Inc.) and the resulting images were analyzed with the manufacturer`s software.
The nucleotide sequence was conceptually translated using the ORF Finder program maintained by the National Center for Biotechnology Information (NCBI) web site (http://www.ncbi.nlm.nih.gov) and the putative ORF encoding a 60-amino acid protein representing catfish MT was identified. Nucleotide and amino acid sequences were subjected to a BLAST search at the NCBI web site to identify previously cloned, related genes and proteins. The derived amino acid sequences of MT were compared using a maximum parsimony tree constructed by a branch and band search algorithm using PAUP (3.1) software (Champaign, IL).
Statistics
Between-group differences were determined by 1-way ANOVA ( = 0.05) followed by the Student-Newman-Kuels test or Bonferroni's Multiple range for significance. Time- and dose-dependent differences were simultaneously evaluated by 2-way ANOVA (Statview 5.1). Correlation analysis was performed by simple linear correlation (
= 0.05).
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RESULTS |
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Liver somatic (LSI) and Condition indices (CI) were significantly affected by treatment. CI in males were significantly reduced after 4, 6 and 8 weeks of treatment with 2.0 mg/L PM (Fig. 5). However, the reduction was not related to time of exposure, since no significant differences in CI were observed between animals sampled at 4, 6, and 8 weeks. LSI was reduced in females at all doses after 6 weeks, but then recovered by 8 weeks (Fig. 6
). A similar trend was observed in males, where reductions in LSI were observed after 4 weeks, then recovered to control levels after 6 weeks with the exception of the 1.0 mg/L animals where LSI was less than controls after 8 weeks (Figure 7
).
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DISCUSSION |
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Since PM is a strong oxidant, putative toxicity to fish would be mediated through adversely affecting the skin or gill (Brouck and Johnson, 1979). In order to assess sublethal damage to the gill, expression of the stress protein metallothionein was examined in gills obtained from animals exposed to PM. In rodents, injection of Mn caused a significant induction of hepatic MT (Waalkes and Klaassen, 1985
), which is not caused by binding of Mn to metal regulatory transcription factors (Bracken and Klaassen 1987
). However, in catfish, no significant increases were observed in gill MT mRNA following 8 weeks of exposure to PM. In fact, significant decreases were observed in the 2.0 mg PM/L concentrations. Several studies have demonstrated piscine MT is induced by other oxidants such as hydrogen peroxide (Kling et al., 1996
; Schlenk and Rice 1998
). The lack of MT induction at the gill and the failure of catfish to accumulate Mn in tissues suggest that Mn, when in the form of potassium permanganate, is not readily absorbed and not bioavailable to the catfish.
In order to measure MT expression, an MT cDNA probe was isolated from a cadmium-treated channel catfish liver. Characteristics of the cDNA are indicative of Class I MT sequences (Kagi and Schaffer 1988). There were 4 amino acid residues that were unique to catfish MT compared to other teleosts (alanine-26; cysteine 27; D-52; K-57). All fish MT sequences that have been identified to date have conserved number and position of cysteines. All fish MTs have 60 amino acids, with the exception of Trout MT-A, which has an additional alanine at the junction between the second and third introns that establishes the boundary between the alpha and beta domains of the protein,
A 50% majority-ruled consensus tree derived from 21 most parsimonious trees is consistent with earlier reports indicating strong sequence homology of MT between teleostian orders (Olsson 1996). Channel catfish are classified in the family Ictaluridae and order Cypriniformes. In only two parsimony trees, did catfish MT fail to cluster with other Cyprinids Stoneloach/Goldfish (data not shown), forming an outgroup to Winter Flounder, Plaice, Cod, Tilapia, Stoneloach, and Goldfish.
Earlier studies with cadmium-treated channel catfish indicated two mRNA transcripts which correlated with two MT proteins that were purified from the livers of cadmium-treated fish (Zhang and Schlenk 1995). The identification of only one RT-PCR product suggests that multiple splice sites may be present in catfish MT mRNA, allowing transcripts of varied length to be formed. The presence of multiple isoforms may also be explained by post-translational modification (i.e., acetylation) of the translated product, which has been shown to occur in other MTs (Roesijadi 1992
). Another possibility is that multiple MT genes may exist. However, this seems unlikely since initial Northern analysis used a Winter flounder cDNA probe indicating similar sequence homology (Zhang and Schlenk 1995
). If another unique MT gene is present in catfish, then the initial 18 nucleotides must be drastically different from any other teleost MT, since the sequence homology for the first 6 amino acids is so highly conserved in fish (Chan 1994
) (Olsson 1996
).
In summary the oxidant, potassium permanganate, did not cause mortality in channel catfish following 8 weeks of intermittent exposure. Significant temporal effects on combined growth, length, and liver weights were observed following 4 weeks of exposure, but returned to baseline by 8 weeks. No relationship was observed between the oxidant-stress protein metallothionein (MT), dose or any whole animal measurement. In order to carry out MT measurements, a single cDNA was isolated by 3`RACE RT-PCR from cadmium-treated livers of channel catfish and was characterized. The structural identification of this product validates earlier studies using RT-PCR to measure MT expression in catfish following treatments with assorted metals.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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