Metallothionein and mercury tolerance in the killifish, fundulus heteroclitus

Synthesis of metallothionein (MT) as a possible mechanism by which Fundulus heteroclitus adapts to heavy metal pollution has been investigated in relation to responses to methylmercuric chloride (meHg) and HgCl₂(Hg²⁺) at different life stages.Clutches of embryos from unpolluted sites exhibit teratogenic responses to a standard dose of meHg or of Hg²⁺ which range from little or no effect (tolerant) to severely affected or almost complete inhibition of development (susceptible). At the time of hatching, untreated embryos from tolerant clutches had twice as much of a protein which co-migrates in SDS-PAGE gels with Fundulus MT as do those of susceptible clutches. This difference could be a mechanism for conveying tolerance to Hg²⁺ and also to meHg if demethylation occurs. However, eggs at the time of deposition had virtually no MT, suggesting that the protein is synthesized too late to provide tolerance in the early stages of development which are critical in the etiology of malformations. Treatment of embryos with non-teratogenic doses of either meHg or Hg²⁺ did not increase the amount of MT-like protein. This may indicate that the thionein gene is already fully active, and would also explain observations that Hg²⁺ pretreatment of embryos does not enhance tolerance to Hg²⁺ in the larval stage.MT, isolated from livers of Hg²⁺- or Cu²⁺-dosed or control adults, is associated with Cu and Zn but not with Hg, although it can bind Hg²⁺in vitro. Fundulus, like other fish, methylates much (ca. 75 %) of its Hg burden, and meHg is considered by some not to react with MT. Fundulus liver has its Hg burden distributed between homogenate sediment (25 %), the cytosol (60 %) and the remainder in lipid. The liver responds to Hg²⁺ treatment by increasing lipid content. This may allow sequestration of some lipid-soluble meHg. Of the cytosolic Hg, most is associated with larger proteins and some (ca. 25 %) is dialyzable.These data suggest that metallothionein is not a significant factor in Hg tolerance in Fundulus adults, and has a questionable role in embryonic and larval tolerance to Hg²⁺ and none to the more toxic meHg.     

Benzo(a)pyrene induced glycine N-methyltransferase messenger RNA expression in Fundulus heteroclitus embryos

Glycine N-methyltransferase (GNMT) is a mediator in the methionine and folate cycles, and is responsible for the transfer of a methyl group from S-adenosylmethionine (SAM) to glycine forming S-adenosylhomocysteine (SAH) and sarcosine. All the known DNA methyltransferases use SAM as a methyl donor thus, GNMT is critically involved in regulation of DNA methylation. Altered GNMT activities have been associated with liver pathologies including hepatocellular carcinoma. The homotetramer form of GNMT is enzymatically active, but the homodimeric form has been suggested as the 4S PAH-binding protein which may mediate CYP1A expression. To further understand the role of GNMT in benzo(a)pyrene (BaP)-related toxicity, full length Fundulus heteroclitus GNMT cDNA was cloned from adult liver. The open reading frame (ORF) of GNMT is 888 base pairs long and encodes a deduced protein of 295 amino acids which has 74% identity with human GNMT. Expression of GNMT mRNA was determined by quantitative RT-PCR. In unfertilized, 2 days postfertilization (dpf), and 3 dpf embryos GNMT was constitutively higher than in 4, 7, 10 or 14 dpf embryos. Embryos were also exposed to waterborne BaP at 10 and 100 μg L⁻¹, and by 10 dpf the higher BaP dose caused increased expression of GNMT mRNA. These results suggest that PAH exposure may alter expression of an important physiological methylation mediator. Future work will be necessary to determine enzyme level effects of BaP exposure as well.     

In vitro mechanistic differences in benzo[a]pyrene–DNA adduct formation using fish liver and mussel digestive gland microsomal activating systems

Turbot (Scophthalmus maximus) and mussel (Mytilus edulis) microsomes were incubated with DNA to examine if microsomal in vitro metabolism of BaP could result in DNA adducts detected by ³²P-postlabelling. Turbot DNA was incubated with benzo[a]pyrene (BaP), NADPH and microsomal activating systems prepared from either livers of unexposed turbot, turbot exposed to BaP or β-naphthoflavone (ß-NF) or digestive glands from mussels. The β-NF activating system generated the highest levels of DNA adducts detected in this study (451.7 adducts per 10⁸ nucleotides) and were distributed in three discrete adduct TLC spots, one of which (97% of the total adducts) co-migrated with the ³²P-postlabelled BaP 7,8-diol, 9,10-epoxide-N²-guanine adduct. Fewer adducts (P <0.05) were generated by BaP-induced microsomes (9.4–30.6 adducts per 10⁸ nucleotides) but levels were higher (P <0.05) than those generated from untreated fish (3.5 adducts per 10⁸ nucleotides). Co-incubation with 500 μM α-naphthoflavone (α-NF) resulted in 97–99% inhibition in adduct formation implicating cytochrome P450-dependent (CYP) bioactivation however there was some evidence for carry over of BaP in the liver microsomal preparations from BaP injected fish. In contrast to the fish activating systems, no DNA adducts were observed when mussel microsomes were incubated with BaP, DNA and NADPH.     

Induction of zona radiata and vitellogenin genes in estradiol and nonylphenol exposed male sheepshead minnows (Cyprinodon variegatus)

Several genes normally induced by estradiol (E₂) in female fish, those for vitellogenins (VTGs) and zona radiata proteins (ZRPs), are also inducible in males exposed to estrogenic chemicals. Male sheepshead minnows (SHM) were exposed to both E₂ and para-nonylphenol (NP), at several doses and times to determine a dose-response. Quantitative real time PCR was used to measure mRNA for VTG1, VTG2, ZRP2 and ZRP3. Both E₂ and NP elicited a dose-response increase in all of the mRNAs tested. Exposure to both chemicals resulted in VTG2 expression at about a 10-fold lower level than VTG1, and ZRP2 expression at a lower level than ZRP3.     

Trophic transfer of benzo[a]pyrene metabolites between benthic marine organisms

This study was undertaken to determine the potential for trophic transfer of polycyclic aromatic hydrocarbon (PAH) metabolites from infaunal organisms to bottom-feeding fish. Winter flounder, Pseudopleuronectes americanus, were given single oral doses of ground polychaetes (Nereis virens), either treated with pure [¹⁴C]henzo[a]pyrene (BaP) or containing a mixture of naturally produced radiolabeled BaP and BaP metabolites. Fish were sacrificed 24 h after feeding and total accumulated radioactivity and metabolite class profiles determined in major organs. Metabolites produced by worms were absorbed by flounder, although as a percentage of dose given they were less available than parent BaP. Comparison of metabolite profiles in the worm diet and in target organs in the fish indicated that metabolites accumulated through the diet can be further modified by the prey organism and can lead to the formation of bound residues. These results demonstrate that PAH metabolites in the diet are available for accumulation. Furthermore, metabolites absorbed appear to be susceptible to metabolic alteration by consumer organisms.     

Benzo[a]pyrene-dione-stimulated oxyradical production by microsomes of digestive gland of the common mussel, Mytilus edulis L

The potential of benzo[ a]pyrene (BaP) quinones (diones) to stimulate NAD(P)H-dependent hydroxyl radical (·OH) production (2-keto-4-methiolbutyric acid (KMBA) oxidation) by digestive gland microsomes of M. edulis was studied using iron/EDTA as a promotor of the Haber-Weiss reaction (O₂⁻ + H₂O₂ = ·OH + OH⁻ + O₂). Stimulation of basal rates of KMBA oxidation was observed for all three diones. Stimulation was similar for the 1,6- and 3,6-diones, but much less for the 6,12-dione, and greater for the NADH than the NADPH-dependent reaction, viz. % increases of 78 to 333 (NADH) compared to 0 to 78 (NADPH). Maximal KMBA oxidation was obtained at dione concentrations of 12.5 to 50 μm. Inhibition of KMBA oxidation by Superoxide dismutase and catalase indicated the involvement of respectively Superoxide anion radical (O₂⁻) and hydrogen peroxide (H₂O₂) in 1,6-dione stimulated NADPH-dependent · OH formation. The apparent K_m values of xenobiotic-stimulated KMBA oxidation were lower for BaP-diones than for the model redox cycling quinone menadione (2-methyl-1,4-naphthoquinone), viz. in μm, 0.6 to 14.6 (NADH) and 4.4 to 28.5 (NADPH) compared to 315 to 457 (NADH) and 72 to 103 (NADPH). The results are consistent with metabolism of BaP to diones and resultant enhanced generation of oxyradicals being a potential mechanism of pollutant-mediated toxicity in molluscs.     

Simultaneous exposure of English sole (Parophrys vetulus) to sediment-associated xenobiotics: Part 2—chronic exposure to an urban estuarine sediment with added 3H-benzo[a]pyrene and 14C-polychlorinated biphenyls

• 1.1. The accumulation of aromatic hydrocarbons (AHs) and polychlorinated biphenyls (PCBs) kv English sole (Parophrys vetulus) exposed for up to 108 days to an urban (test) sediment was compared to that for English sole exposed to sediment from a reference area. The concentrations of identified AHs and PCBs in the test sediment were 32 μg/g dry weight and 2·2 μg/g dry weight, respectively. English sole exposed to test and reference sediments for 108 days had biliary concentrations of aromatic compounds fluorescing at BaP wavelengths of 0·6 μg/g, wet weight and hepatic concentrations of PCBs of 1·4 ± 0·6 μg/g wet weight which were ten and eight times, respectively, as great as those in reference sole. These results show that accumulation of AHs and PCBs from sediment by English sole is a significant route of uptake in contaminated environments.
• 2.2. ³H-benzo(a)pyrene (BaP) and ¹⁴C-PCBs were added to the test sediment to compare the uptake and metabolism of BaP to that of PCBs by sole. Steady-state concentrations of ³H-BaP and ¹⁴C-PCBs in tissues and fluids were reached by 4 days and 14 days, respectively. At steady state, the level of covalent binding of ³H-BaP to hepatic proteins in test sole was six times greater than that of ¹⁴C-PCBs, and there was significantly greater relative formation of BaP-glutathione (GSH) conjugates than PCB-GSH conjugates. Thus, the amount of BaP metabolized to reactive metabolites was greater than that for PCBs, even though the concentration of ¹⁴C-PCBs in liver of test sole was five times as great as that of ³H-BaP.
• 3.3. Trace amounts of ³H-BaP were also added to the reference sediment to determine how simultaneous exposure of English sole to those contaminants already present in the test sediment affected the metabolism and disposition of BaP. The ³H-BaP concentration ratios for liver to sediment and bile to sediment, as well as proportions of biliary BaP-GSH conjugates in test sole, were significantly greater than in reference sole. Thus, the formation and accumulation of potentially toxic metabolites of a carcinogenic AH (BaP) in sole liver were increased by simultaneous exposure to contaminants present in an urban sediment.

     

Effects of benzo[a]pyrene on DNA damage and histological alterations in gonad of scallop Chlamys farreri

To investigate the biological impact of polycyclic aromatic hydrocarbons (PAHs) on reproductive system, scallops Chlamys farreri were continuously exposed to benzo[a]pyrene (BaP) (0.5, 3, 10 μg L^?1) during 15 days. DNA damage and histological alterations were examined in female gonad. DNA strand break levels significantly increased after 12 h exposure, and remained high and significantly different from control values until the end of the exposure. In the ovaries of the scallops exposed to 10 μg L^?1 BaP for ten days, histological analysis showed that the cytoplasts of the oocytes in the outer layer of the ovaries became sparse, and the nuclear membranes were obscure. Damaging effects on ovaries and oocytes were more severe after 15 days exposure. Degenerating oocytes increased and the connective tissue of the ovary envelops became loose. Electron microscopic examination revealed ultrastructural aspects of degenerating oocyte and degenerated oocyte after 15 days exposure.     

Accumulation of total CO2 during stagnation in the Baltic Sea deep water and its relationship to nutrient and oxygen concentrations

Accumulation of total CO₂ (C_T) was investigated in the sub-halocline deep water of the Gotland Sea (Baltic Sea) during a period of stagnation from 1995 to 1999. Depth profiles for C_T, nitrate, phosphate, and oxygen were measured during seven cruises in a grid consisting of 26 stations. The mean C_T increased by more than 60 μmol/kg from October 1995 to July 1999 corresponding to a mean accumulation rate of 1.1 mol/m² year. Taking into account vertical mixing, the vertical distribution of the C_T accumulation was used to determine mineralization rates at different depths. High rates immediately below the halocline indicated the existence of a fraction of organic matter, which is rapidly mineralized during sinking through the water column. A second fraction is more refractive and accumulates at the sediment surface in the deep center of the basin where it is slowly mineralized and partly buried. Phosphate release rates in anoxic waters and especially at the redoxcline were substantially higher than those estimated on the basis of the carbon mineralization and the Redfield C/P ratio. This is attributed to non-Redfield mineralization ratios and the dissolution of iron oxide/phosphate associates. The formation of nitrate by mineralization under oxic conditions was almost completely compensated by denitrification. Using the carbon mineralization rates and a C/N ratio of 8.4, a denitrification rate of 280 mmol/m² year was obtained, which approximately balances the input of nitrate/ammonia into the surface water. Relating the apparent oxygen utilization (AOU) to the C_T fraction that was generated by mineralization yielded a carbon mineralization/O₂ consumption ratio of 0.83.     

富油微藻富碳培养下油脂积累及固碳研究

微藻固碳是一种新型节能减排技术,具有长期可持续发展的潜力。本文对两株富油微藻（球等鞭金藻和微拟球藻）进行了富碳培养下生长特性及中性脂积累特性的研究。两株富油微藻的最佳培养条件为10%CO₂浓度和f培养基。本研究对两株富油微藻的最大生物量产率、总脂含量、最大油脂产率、微藻的C含量和CO₂固定率进行了测定。球等鞭金藻的各参数指标分别为：142.42±4.58g/（m²·d）,39.95%±0.77%,84.47±1.56g/（m²·d）,45.98%±1.75%和33.74±1.65g/（m²·d）。微拟球藻的各参数指标分别为：149.92±1.80g/（m²·d）,37.91%±0.58%,89.90±1.98g/（m²·d）,46.88%±2.01%和34.08±1.32g/（m²·d）。实验结果显示,两株海洋微藻均属于高固碳优良藻株,适合应用于微藻烟气减排技术开发,具备用于海洋生物质能耦合CO₂减排开发的潜力。     

Effects of the inhibitor ellipticine on cytochrome P450-reductase and cytochrome P450 (1A) function in hepatic microsomes of flounder (Platichthys flesus)

The effects of the mammalian inhibitor ellipticine (5,11-dimethyl-[6H]-pyrido[4,3b] carbazole) were examined in a mechanistic study of the cytochrome P450 monooxygenase system of control and β-naphthoflavone (βNF)-induced hepatic microsomes of Platichthys flesus. Ellipticine was indicated to bind to the haem moiety of cytochrome P450s (gave type II binding spectra) and to inhibit the transfer of electrons from both the hydrophobic binding site of cytochrome P450 reductase (P450R) to P450 (inhibited P450R reductase activity) and the hydrophilic binding site of P450R to soluble electron acceptors (inhibited NAD(P)H-cytochrome c reductase activity). No effect was seen on cytochrome b₅ reductase activity. Ellipticine inhibition indicated the involvement of (i) P450R (possibly also P450s) in NADPH- but not NADH- dependent hydroxyl radical production, and (ii) electron transfer and P450/P450R interaction in NADPH-dependent cytochrome P450 1A-catalysed monooxygenation (7-ethoxyresorufin O-deethylase activity and benzo(a)pyrene (BaP) metabolism). Differential effects of ellipticine on cumene hydroperoxide (CHP)-dependent BaP metabolism (P450 peroxidase activity) with CHP concentration indicated the existence of at least two forms of P450 with different substrate affinities for CHP, and different mechanisms of formation for protein adducts and free metabolites. Overall, the studies indicate the primary site of action of ellipticine in P. flesus is binding between Fe³⁺-P450 and P450R.     

Role of the mucous surface coat, dietary bulk and mucosal cell turnover in the intestinal disposition of benzo(a)pyrene (BaP)

The dispositon of ³H-benzo(a)pyrene (BaP) equivalents from the diet was examined at the lumen-mucosal cell interface of the proximal, medial and distal regions of the catfish intestine. ³H-BaP (500 μd, 20 μg/kg) was administered via gavage in maintenance diet to two groups of catfish. One group was fed daily after dosing while the other was fasted until collection of intestinal tissues. Autoradiography of cryosectioned tissues and computer-enhanced image analysis allowed examination of BaP disposition. BaP was found to selectively localize at high concentrations in the mucous surface coat and corresponding villi of the colon. Supplemental feeding of uncontaminated diet lowered BaP concentrations in these regions. In a similar but separate experiment, ³H-thymidine (666 μCi) was administered to two groups of catfish by i.p. injection to examine mucosal cell turnover in relation to BaP turnover. ³H-Thymidine incorporation was greatest in the distal regions of both fasted and fed groups. Thymidine dynamics indicated that feeding was related to only marginal changes in cell turnover, which suggests that losses in intravillus BaP with feeding may not be wholly explained by intestinal cell turnover. Results from this study suggest that the mucous surface coat of the intestine is a major factor in the regional disposition of dietary carcinogens and removal of BaP from this mucin layer is related to the intake of dietary bulk.     

Effect of varying sediment organic carbon content upon the dermal bioavailability and disposition of benzo(a)pyrene in the catfish, Ictalurus punctatus

The dermal absorption of ³H-benzo(a) pyrene (BaP) from sediments of varying organic carbon content was examined in the catfish (Ictalurus punctatus). Test sediments of differing organic carbon concentrations were formulated from natural sediments with the addition of quartz sand to provide organic carbon content of 1.8, 3.7 and 6.1%. Sediments (1.0 g dry wt), spiked with BaP to provide 20.4 μCi at a BaP dose of 75 ng/g, were applied for 6 h to the skin surface (12 cm²) of anesthetized fish. Dermal exposure to BaP in formulated sediments at 6.1, 3.7 and 1.8% resulted in 6-h mass balance bioavailability values of 19.4, 21.9 and 23.2%, respectively. In all treatments, skin in direct contact with sediments had the highest concentration followed by the corresponding muscle layer. Muscle and skin not in direct contact with the labeled sediment had values which averaged 7–580 times lower than the corresponding exposed tissues. Other tissue values ranged from 0.44 pg/g in the heart to 64.21 pg/g in blood. These findings demonstrate appreciable uptake of BaP from surface applied sediments. Within the range examined, varying organic carbon content had a small, but inverse effect upon the dermal bioavailability of BaP.     

Inhibition of essential amino acid absorption in marine fishes by mercury

In polluted coastal waters marine fish can concentrate heavy metals in the intestine two or more orders of magnitude above those found in the water column and several-fold higher than the sediment level. These accumulations are primarily due to ingestion of contaminated food and drink. Relatively long residence time of gut content, low permeability coefficient of divalent cations through the lipid bilayers of membranes and profuse binding to the negative charge sites on the mucosal side of the gut, all combine to increase the concentration of heavy metals in the lumen. Do these high levels of heavy metals interfere with the normal processes of nutrient absorption? We have measured the effect of HgCl₂ and CH₃HgCl on the absorption of the essential amino acids, l-leucine, l-isoleucine, l-lysine and l-methionine, by the intestine of Fundulus and toadfish in vitro and in vivo. At concentrations expected in the gut, HgCl₂ can inhibit 20–80% of the leucine absorption; CH₃HgCl is less potent in this respect. While the action of Hg²⁺ appears to be limited to the luminal side, the more lipophilic CH₃Hg⁺ rapidly penetrates the epithelial cell layer and can inhibit the intracellular energy production needed for transport. The implications of these findings for growth and survival of marine fishes are discussed.     

海马齿对不同盐度水质的碳汇作用以及不同形态氮利用的研究

为探讨海马齿(Sesuvium portulacastrum)对水域环境修复作用,本文研究了水培海马齿对不同盐度水质的碳汇作用以及不同形态氮的利用情况。实验设计0、10、20、30、35盐度梯度,海马齿水培时间82 d,然后测定植株干重、营养元素含量以及积累速率,最后在抑菌与不抑菌条件下研究海马齿根际与铵态氮(NH₄⁺-N)、硝态氮(NO₃^--N)、无机磷(PO₄^3-)以及色氨酸(Trp)吸收转化关系。研究结果表明,盐度10条件下海马齿植株干重、有机元素含量以及积累速率最高,有机碳、有机氮与有机磷积累速率分别为(5.572±1.611)、(0.313±0.058)、(0.057±0.013)mg/(d·ind.),而高盐环境35盐度条件下对海马齿生长造成一定胁迫。盐度0～35范围,海马齿均未出现死亡现象。不同盐度抑菌培养条件下,色氨酸与无机氮共存时均能被能被海马齿利用,色氨酸利用量远高于硝态氮、铵态氮;不抑菌条件下铵态氮则表现出增加的结果。海马齿作用在盐度...     

Behavior and brain chemistry correlates in mummichogs (Fundulus heteroclitus) from polluted and unpolluted environments

The ability of Fundulus from a polluted environment to capture a prey organism, the grass shrimp (Palaemonetes pugio), was compared to that of Fundulus from an unpolluted reference site. Fish from the unpolluted site took significantly more prey (p < 0.1) during the first 5–10 min of each trial than fish from the polluted site.Analysis of the neurotransmitters dopamine (DA) and serotonin (5-HT) and their metabolites, DOPAC, homovanillic acid (HVA), and 5-hydroxy-indolacetic acid (5-HIAA) in cerebellums and medullas was performed by HPLC. Mummichogs from the polluted site had significantly lower concentrations of 5-HT and its metabolite 5-HIAA in their medullas, but not cerebellums, than fish from the reference site (p < 0.05). Concentrations of DA, DOPAC and HVA were not different between populations in either part of the brain. Neurotransmitter concentrations were shown to be independent of size and therefore, presumably, age.Analysis of Hg in livers and brains of these mummichogs by atomic absorption spectrophotometry showed significant differences between the two populations for both organs. Earlier work showed that the behavioral deficit could be elicited by Hg; thus, this was the putative neurotoxic agent. However, there are other candidates which may contribute to neurotoxicity in the study site.