Civa Klorür Uygulanan Gökkuşağı Alabalıkları (Oncorhynchus Mykiss)’ nın Solungaç Dokusunda Nitrozatif ve Oksidatif Stresin Rolü
Yıl 2021,
, 957 - 962, 31.10.2021
Mehmet Reşit Taysı
,
Muammer Kırıcı
,
Mahinur Kırıcı
,
Bünyamin Sögüt
,
Mehmet Akif Bozdayı
,
Mehmet Tarakçıoğlu
Seyithan Taysı
Öz
Çalışmanın amacı, civa klorürün (HgCl2) 59,43 ± 7,21 g ağırlığındaki gökkuşağı alabalıklarında (Oncorhynchus mykiss) toksik etkisinin ve neden olduğu oksidatif stresin dinamiklerini belirlemektir. Bu amaçla, her grupta 10 balık (n = 10) olacak şekilde 4 grupta toplam 40 balık, LD50'nin %25 ve %50'sine (137,75 μg/L ve 275 μg/L) 2 ve 7 gün süreyle maruz bırakıldı. Oksidatif / nitrozatif stresi belirlemek için, Peroksinitrit (ONOOˉ mmol / L), Toplam Oksidan Seviyesi (TOS mmol H2O2 Eq / L), Toplam Antioksidan Seviyesi (TAS mmol Trolox Eq / L), OSI (TOS / TASx10) ve Malondialdehit (MDA) seviyesi belirlendi. Solungaç dokularında TAS, OSI ve MDA düzeyleri açısından gruplar arasındaki fark istatistiksel olarak anlamlı iken (P <0.05), bu fark ONOOˉ ve TOS değerleri açısından istatistiksel olarak önemsiz çıktı (P> 0.05). Sonuç olarak, HgCl2 solungaç dokusunda stres göstergeleri olan ONOOˉ, OSI ve MDA seviyelerini arttırdığı için strese neden olduğu ve balıklar için toksik olduğu görüldü.
Kaynakça
- Ahlatci A, Kuzhan A, Taysi S, Demirtas OC, Alkis HE, Tarakcioglu M, Demirci A, Caglayan D, Saricicek E, Cinar K 2014. Radiation-modifying abilities of Nigella sativa and thymoquinone on radiation-induced nitrosative stress in the brain tissue. Phytomedicine 21:740-744.
- Al-Nimer MS, Al-Ani FS, Ali FS 2012. Role of nitrosative and oxidative stress in neuropathy in patients with type 2 diabetes mellitus. J Neurosci Rural Pract 3:41-44.
- Bagchi D, Bagchi M, Hassoun EA, Stohs SJ 1995. In vitro and in vivo generation of reactive oxygen species, DNA damage and lactate dehydrogenase leakage by selected pesticides. Toxicology 104:129-140.
- Berntssen MHG, Aatland A, Handy RD 2003. Chronic dietary mercury exposure causes oxidative stress, brain lesions, and altered behaviour in Atlantic salmon (Salmo salar) parr. Aquat Toxicol 65:55-72.
- Bollen A, Wenke A, Biester H 2008. Mercury speciation analyses in HgCl(2)-contaminated soils and groundwater--implications for risk assessment and remediation strategies. Water Res 42:91-100.
- Brandao F, Cappello T, Raimundo J, Santos MA, Maisano M, Mauceri A, Pacheco M, Pereira P 2015. Unravelling the mechanisms of mercury hepatotoxicity in wild fish (Liza aurata) through a triad approach: bioaccumulation, metabolomic profiles and oxidative stress. Metallomics 7:1352-1363.
- Cappello T, Brandao F, Guilherme S, Santos MA, Maisano M, Mauceri A, Canario J, Pacheco M, Pereira P 2016a. Insights into the mechanisms underlying mercury-induced oxidative stress in gills of wild fish (Liza aurata) combining H-1 NMR metabolomics and conventional biochemical assays. Sci Total Environ 548:13-24.
- Cappello T, Pereira P, Maisano M, Mauceri A, Pacheco M, Fasulo S 2016b. Advances in understanding the mechanisms of mercury toxicity in wild golden grey mullet (Liza aurata) by (1)H NMR-based metabolomics. Environ Pollut 219:139-148.
- Clarkson TW, Magos L 2006. The toxicology of mercury and its chemical compounds. Crit Rev Toxicol 36:609-662.
- Crocker IP, Kenny LC, Thornton WA, Baker PN 2005. Excessive stimulation of poly(ADP-ribosyl)ation contributes to endothelial dysfunction in pre-eclampsia. Brit J Pharmacol 144:772-780.
- De Domenico E, Mauceri A, Giordano D, Maisano M, Gioffre G, Natalotto A, D'Agata A, Ferrante M, Brundo MV, Fasulo S 2011. Effects of "in vivo" exposure to toxic sediments on juveniles of sea bass (Dicentrarchus labrax). Aquat Toxicol 105:688-697.
- Dean RJ, Shimmield TM, Black KD 2007. Copper, zinc and cadmium in marine cage fish farm sediments: an extensive survey. Environ Pollut 145:84-95.
- Devlin EW 2006. Acute toxicity, uptake and histopathology of aqueous methyl mercury to fathead minnow embryos. Ecotoxicology 15:97-110.
- Echeverria-Saenz S, Mena F, Arias-Andres M, Vargas S, Ruepert C, Van den Brink PJ, Castillo LE, Gunnarsson JS 2018. In situ toxicity and ecological risk assessment of agro-pesticide runoff in the Madre de Dios River in Costa Rica. Environ Sci Pollut R 25:13270-13282.
- Erel O 2004. A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical cation. Clin Biochem 37:277-285.
- Erel O 2005. A new automated colorimetric method for measuring total oxidant status. Clin Biochem 38:1103-1111.
- Erickson RJ, Nichols JW, Cook PM 2008. Bioavailability of chemical contaminants in aquatic systems, In: Di Giulio RT, Hinton DE. In: The Toxicology of Fishes. Florida, USA: CRC Press. p 9-45.
- Ertosun FM, S. Z, B. C, H. U, A. Y, S. T 2020. Reactive Oxygen Species Dr. 2020. Increased Nitrosative Stress and Oxidative DNA Damage in Patients with Carbon Monoxide Poisoning. Reactive Oxygen Species 9:136-143.
- Fasulo S, Marino S, Mauceri A, Maisano M, Giannetto A, D'Agata A, Parrino V, Minutoli R, De Domenico E 2010. A multibiomarker approach in Coris julis living in a natural environment. Ecotoxicol Environ Saf 73:1565-1573.
- Gammons CH, Slotton DG, Gerbrandt B, Weight W, Young CA, McNearny RL, Camac E, Calderon R, Tapia H 2006. Mercury concentrations of fish, river water, and sediment in the Rio Ramis-Lake Titicaca watershed, Peru. Sci Total Environ 368:637-648.
- Giari L, Simoni E, Manera M, Dezfuli BS 2008. Histo-cytological responses of Dicentrarchus labrax (L.) following mercury exposure. Ecotoxicol Environ Saf 70:400-410.
- Guardiola FA, Chaves-Pozo E, Espinosa C, Romero D, Meseguer J, Cuesta A, Esteban MA 2016. Mercury Accumulation, Structural Damages, and Antioxidant and Immune Status Changes in the Gilthead Seabream (Sparus aurata L.) Exposed to Methylmercury. Arch Environ Contam Toxicol 70:734-746.
- Hopkins WA, Tatara CP, Brant HA, Jagoe CH 2003. Relationships between mercury body concentrations, standard metabolic rate, and body mass in eastern mosquitofish (Gambusia holbrooki) from three experimental populations. Environ Toxicol Chem 22:586-590.
- Jagoe CH, Faivre A, Newman MC 1996. Morphological and morphometric changes in the gills of mosquitofish (Gambusia holbrooki) after exposure to mercury (II). Aquat Toxicol 34:163-183.
- Jiang F, Liu J, Zeng XY, Yu LQ, Liu CS, Wang J 2018. Tris (2-butoxyethyl) phosphate affects motor behavior and axonal growth in 1 zebrafish (Danio rerio) larvae. Aquat Toxicol 198:215-223.
- Kirici M, Kirici M, Beydemir S, Atamanalp M 2016. Purification of Carbonic Anhydrase from Capoeta umbla (Heckel, 1843) Gills and Toxicological Effects of Some Metals on Enzyme Activity. Turk J Fish Aquat Sc 16:169-175.
- Lima APS, Sarkis JES, Shihomatsu HM, Muller RCS 2005. Mercury and selenium concentrations in fish samples from Cachoeira do PiriaMunicipality, ParaState, Brazil. Environ Res 97:236-244.
- Lungu-Mitea S, Oskarsson A, Lundqvist J 2018. Development of an oxidative stress in vitro assay in zebrafish (Danio rerio) cell lines. Sci Rep-Uk 8.
- Matos RC, Vieira C, Morais S, Pereira ML, Pedrosa J 2010. Toxicity of chromated copper arsenate: a study in mice. Environ Res 110:424-427.
- Mauceri A, Fossi MC, Leonzio C, Ancora S, Minniti F, Maisano M, Lo Cascio P, Ferrando S, Fasulo S 2005. Stress factors in the gills of Liza aurata (Perciformes, Mugilidae) living in polluted environments. Ital J Zool 72:285-292.
- McRae NK, Glover CN, Burket SR, Brooks BW, Gaw S 2018. Acute Exposure to an Environmentally Relevant Concentration of Diclofenac Elicits Oxidative Stress in the Culturally Important Galaxiid Fish Galaxias maculatus. Environ Toxicol Chem 37:224-235.
- Mieiro CL, Ahmad I, Pereira ME, Duarte AC, Pacheco M 2010. Antioxidant system breakdown in brain of feral golden grey mullet (Liza aurata) as an effect of mercury exposure. Ecotoxicology 19:1034-1045.
- Monteiro DA, Rantin FT, Kalinin AL 2013a. Dietary intake of inorganic mercury: bioaccumulation and oxidative stress parameters in the neotropical fish Hoplias malabaricus. Ecotoxicology 22:446-456.
- Monteiro DA, Thomaz JM, Rantin FT, Kalinin AL 2013b. Cardiorespiratory responses to graded hypoxia in the neotropical fish matrinxa (Brycon amazonicus) and traira (Hoplias malabaricus) after waterborne or trophic exposure to inorganic mercury. Aquat Toxicol 140-141:346-355.
- Ohkawa H, Ohishi N, Yagi K 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95:351-358.
- Oliveira Ribeiro CA, Pelletier E, Pfeiffer WC, Rouleau C 2000. Comparative uptake, bioaccumulation, and gill damages of inorganic mercury in tropical and nordic freshwater fish. Environ Res 83:286-292.
- Perottoni J, Lobato LP, Silveira A, Batista J, Rocha T, Emanuelli T 2004. Effects of mercury and selenite on delta-aminolevulinate dehydratase activity and on selected oxidative stress parameters in rats. Environ Res 95:166-173.
- Rather MA, Bhat IA, Sharma N, Sharma R 2018. Molecular and Cellular Toxicology of Nanomaterials with Related to Aquatic Organisms. Adv Exp Med Biol 1048:263-284.
- Smith KL, Galloway TS, Depledge MH 2000. Neuro-endocrine biomarkers of pollution-induced stress in marine invertebrates. Sci Total Environ 262:185-190.
- Soares A, Guieysse B, Jefferson B, Cartmell E, Lester JN 2008. Nonylphenol in the environment: A critical review on occurrence, fate, toxicity and treatment in wastewaters. Environ Int 34:1033-1049.
- Szabo C 1998. Role of poly(ADP-ribose)synthetase in inflammation. Eur J Pharmacol 350:1-19.
- Vanuffelen BE, Van Der Zee J, De Koster BM, Vansteveninck J, Elferink JG 1998. Intracellular but not extracellular conversion of nitroxyl anion into nitric oxide leads to stimulation of human neutrophil migration. Biochem J 330 ( Pt 2):719-722.
- Vicari T, Dagostim AC, Klingelfus T, Galvan GL, Monteiro PS, Pereira LD, de Assis HCS, Cestari MM 2018. Co-exposure to titanium dioxide nanoparticles (NpTiO2) and lead at environmentally relevant concentrations in the Neotropical fish species Hoplias intermedius. Toxicology Reports 5:1032-1043.
- Zhang MZ, Li M, Wang RX, Qian YX 2018. Effects of acute ammonia toxicity on oxidative stress, immune response and apoptosis of juvenile yellow catfish Pelteobagrus fulvidraco and the mitigation of exogenous taurine. Fish Shellfish Immun 79:313-320.
The Role of Nitrosative and Oxidative Stress in Rainbow Trout (Oncorhynchus Mykiss) Gill Tissue Applying Mercury Chloride
Yıl 2021,
, 957 - 962, 31.10.2021
Mehmet Reşit Taysı
,
Muammer Kırıcı
,
Mahinur Kırıcı
,
Bünyamin Sögüt
,
Mehmet Akif Bozdayı
,
Mehmet Tarakçıoğlu
Seyithan Taysı
Öz
The aim of the study was to determine the toxic effect and the dynamics of oxidative stress caused by mercury chloride (HgCl2) in rainbow trout (Oncorhynchus mykiss) weighing 59.43 ± 7.21 g.For this purpose, a total of 40 fish in 4 groups, 10 fish in each group (n = 10), were exposed to 25% and 50% (137.75 μg/L and 275 μg/L) of the LD50 for 2 and 7 days. To determine the oxidative/nitrosative stress, Peroxynitrite (ONOOˉ mmol/L), Total Oxidant Level (TOS mmol H2O2 Eq/L), Total Antioxidant Level (TAS mmol Trolox Eq/L), OSI (TOS/TASx10) and Malondialdehyde (MDA) level were determined. While the difference between the groups in terms of TAS, OSI and MDA levels in gill tissues was statistically significant (P<0.05), this difference was statistically insignificant (P>0.05) in terms of ONOOˉ and TOS values. As a result, HgCl2 was found to cause stress and toxic to fish as it increases the levels of ONOOˉ, OSI and MDA, which are indicators of stress in gill tissue.
Kaynakça
- Ahlatci A, Kuzhan A, Taysi S, Demirtas OC, Alkis HE, Tarakcioglu M, Demirci A, Caglayan D, Saricicek E, Cinar K 2014. Radiation-modifying abilities of Nigella sativa and thymoquinone on radiation-induced nitrosative stress in the brain tissue. Phytomedicine 21:740-744.
- Al-Nimer MS, Al-Ani FS, Ali FS 2012. Role of nitrosative and oxidative stress in neuropathy in patients with type 2 diabetes mellitus. J Neurosci Rural Pract 3:41-44.
- Bagchi D, Bagchi M, Hassoun EA, Stohs SJ 1995. In vitro and in vivo generation of reactive oxygen species, DNA damage and lactate dehydrogenase leakage by selected pesticides. Toxicology 104:129-140.
- Berntssen MHG, Aatland A, Handy RD 2003. Chronic dietary mercury exposure causes oxidative stress, brain lesions, and altered behaviour in Atlantic salmon (Salmo salar) parr. Aquat Toxicol 65:55-72.
- Bollen A, Wenke A, Biester H 2008. Mercury speciation analyses in HgCl(2)-contaminated soils and groundwater--implications for risk assessment and remediation strategies. Water Res 42:91-100.
- Brandao F, Cappello T, Raimundo J, Santos MA, Maisano M, Mauceri A, Pacheco M, Pereira P 2015. Unravelling the mechanisms of mercury hepatotoxicity in wild fish (Liza aurata) through a triad approach: bioaccumulation, metabolomic profiles and oxidative stress. Metallomics 7:1352-1363.
- Cappello T, Brandao F, Guilherme S, Santos MA, Maisano M, Mauceri A, Canario J, Pacheco M, Pereira P 2016a. Insights into the mechanisms underlying mercury-induced oxidative stress in gills of wild fish (Liza aurata) combining H-1 NMR metabolomics and conventional biochemical assays. Sci Total Environ 548:13-24.
- Cappello T, Pereira P, Maisano M, Mauceri A, Pacheco M, Fasulo S 2016b. Advances in understanding the mechanisms of mercury toxicity in wild golden grey mullet (Liza aurata) by (1)H NMR-based metabolomics. Environ Pollut 219:139-148.
- Clarkson TW, Magos L 2006. The toxicology of mercury and its chemical compounds. Crit Rev Toxicol 36:609-662.
- Crocker IP, Kenny LC, Thornton WA, Baker PN 2005. Excessive stimulation of poly(ADP-ribosyl)ation contributes to endothelial dysfunction in pre-eclampsia. Brit J Pharmacol 144:772-780.
- De Domenico E, Mauceri A, Giordano D, Maisano M, Gioffre G, Natalotto A, D'Agata A, Ferrante M, Brundo MV, Fasulo S 2011. Effects of "in vivo" exposure to toxic sediments on juveniles of sea bass (Dicentrarchus labrax). Aquat Toxicol 105:688-697.
- Dean RJ, Shimmield TM, Black KD 2007. Copper, zinc and cadmium in marine cage fish farm sediments: an extensive survey. Environ Pollut 145:84-95.
- Devlin EW 2006. Acute toxicity, uptake and histopathology of aqueous methyl mercury to fathead minnow embryos. Ecotoxicology 15:97-110.
- Echeverria-Saenz S, Mena F, Arias-Andres M, Vargas S, Ruepert C, Van den Brink PJ, Castillo LE, Gunnarsson JS 2018. In situ toxicity and ecological risk assessment of agro-pesticide runoff in the Madre de Dios River in Costa Rica. Environ Sci Pollut R 25:13270-13282.
- Erel O 2004. A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical cation. Clin Biochem 37:277-285.
- Erel O 2005. A new automated colorimetric method for measuring total oxidant status. Clin Biochem 38:1103-1111.
- Erickson RJ, Nichols JW, Cook PM 2008. Bioavailability of chemical contaminants in aquatic systems, In: Di Giulio RT, Hinton DE. In: The Toxicology of Fishes. Florida, USA: CRC Press. p 9-45.
- Ertosun FM, S. Z, B. C, H. U, A. Y, S. T 2020. Reactive Oxygen Species Dr. 2020. Increased Nitrosative Stress and Oxidative DNA Damage in Patients with Carbon Monoxide Poisoning. Reactive Oxygen Species 9:136-143.
- Fasulo S, Marino S, Mauceri A, Maisano M, Giannetto A, D'Agata A, Parrino V, Minutoli R, De Domenico E 2010. A multibiomarker approach in Coris julis living in a natural environment. Ecotoxicol Environ Saf 73:1565-1573.
- Gammons CH, Slotton DG, Gerbrandt B, Weight W, Young CA, McNearny RL, Camac E, Calderon R, Tapia H 2006. Mercury concentrations of fish, river water, and sediment in the Rio Ramis-Lake Titicaca watershed, Peru. Sci Total Environ 368:637-648.
- Giari L, Simoni E, Manera M, Dezfuli BS 2008. Histo-cytological responses of Dicentrarchus labrax (L.) following mercury exposure. Ecotoxicol Environ Saf 70:400-410.
- Guardiola FA, Chaves-Pozo E, Espinosa C, Romero D, Meseguer J, Cuesta A, Esteban MA 2016. Mercury Accumulation, Structural Damages, and Antioxidant and Immune Status Changes in the Gilthead Seabream (Sparus aurata L.) Exposed to Methylmercury. Arch Environ Contam Toxicol 70:734-746.
- Hopkins WA, Tatara CP, Brant HA, Jagoe CH 2003. Relationships between mercury body concentrations, standard metabolic rate, and body mass in eastern mosquitofish (Gambusia holbrooki) from three experimental populations. Environ Toxicol Chem 22:586-590.
- Jagoe CH, Faivre A, Newman MC 1996. Morphological and morphometric changes in the gills of mosquitofish (Gambusia holbrooki) after exposure to mercury (II). Aquat Toxicol 34:163-183.
- Jiang F, Liu J, Zeng XY, Yu LQ, Liu CS, Wang J 2018. Tris (2-butoxyethyl) phosphate affects motor behavior and axonal growth in 1 zebrafish (Danio rerio) larvae. Aquat Toxicol 198:215-223.
- Kirici M, Kirici M, Beydemir S, Atamanalp M 2016. Purification of Carbonic Anhydrase from Capoeta umbla (Heckel, 1843) Gills and Toxicological Effects of Some Metals on Enzyme Activity. Turk J Fish Aquat Sc 16:169-175.
- Lima APS, Sarkis JES, Shihomatsu HM, Muller RCS 2005. Mercury and selenium concentrations in fish samples from Cachoeira do PiriaMunicipality, ParaState, Brazil. Environ Res 97:236-244.
- Lungu-Mitea S, Oskarsson A, Lundqvist J 2018. Development of an oxidative stress in vitro assay in zebrafish (Danio rerio) cell lines. Sci Rep-Uk 8.
- Matos RC, Vieira C, Morais S, Pereira ML, Pedrosa J 2010. Toxicity of chromated copper arsenate: a study in mice. Environ Res 110:424-427.
- Mauceri A, Fossi MC, Leonzio C, Ancora S, Minniti F, Maisano M, Lo Cascio P, Ferrando S, Fasulo S 2005. Stress factors in the gills of Liza aurata (Perciformes, Mugilidae) living in polluted environments. Ital J Zool 72:285-292.
- McRae NK, Glover CN, Burket SR, Brooks BW, Gaw S 2018. Acute Exposure to an Environmentally Relevant Concentration of Diclofenac Elicits Oxidative Stress in the Culturally Important Galaxiid Fish Galaxias maculatus. Environ Toxicol Chem 37:224-235.
- Mieiro CL, Ahmad I, Pereira ME, Duarte AC, Pacheco M 2010. Antioxidant system breakdown in brain of feral golden grey mullet (Liza aurata) as an effect of mercury exposure. Ecotoxicology 19:1034-1045.
- Monteiro DA, Rantin FT, Kalinin AL 2013a. Dietary intake of inorganic mercury: bioaccumulation and oxidative stress parameters in the neotropical fish Hoplias malabaricus. Ecotoxicology 22:446-456.
- Monteiro DA, Thomaz JM, Rantin FT, Kalinin AL 2013b. Cardiorespiratory responses to graded hypoxia in the neotropical fish matrinxa (Brycon amazonicus) and traira (Hoplias malabaricus) after waterborne or trophic exposure to inorganic mercury. Aquat Toxicol 140-141:346-355.
- Ohkawa H, Ohishi N, Yagi K 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95:351-358.
- Oliveira Ribeiro CA, Pelletier E, Pfeiffer WC, Rouleau C 2000. Comparative uptake, bioaccumulation, and gill damages of inorganic mercury in tropical and nordic freshwater fish. Environ Res 83:286-292.
- Perottoni J, Lobato LP, Silveira A, Batista J, Rocha T, Emanuelli T 2004. Effects of mercury and selenite on delta-aminolevulinate dehydratase activity and on selected oxidative stress parameters in rats. Environ Res 95:166-173.
- Rather MA, Bhat IA, Sharma N, Sharma R 2018. Molecular and Cellular Toxicology of Nanomaterials with Related to Aquatic Organisms. Adv Exp Med Biol 1048:263-284.
- Smith KL, Galloway TS, Depledge MH 2000. Neuro-endocrine biomarkers of pollution-induced stress in marine invertebrates. Sci Total Environ 262:185-190.
- Soares A, Guieysse B, Jefferson B, Cartmell E, Lester JN 2008. Nonylphenol in the environment: A critical review on occurrence, fate, toxicity and treatment in wastewaters. Environ Int 34:1033-1049.
- Szabo C 1998. Role of poly(ADP-ribose)synthetase in inflammation. Eur J Pharmacol 350:1-19.
- Vanuffelen BE, Van Der Zee J, De Koster BM, Vansteveninck J, Elferink JG 1998. Intracellular but not extracellular conversion of nitroxyl anion into nitric oxide leads to stimulation of human neutrophil migration. Biochem J 330 ( Pt 2):719-722.
- Vicari T, Dagostim AC, Klingelfus T, Galvan GL, Monteiro PS, Pereira LD, de Assis HCS, Cestari MM 2018. Co-exposure to titanium dioxide nanoparticles (NpTiO2) and lead at environmentally relevant concentrations in the Neotropical fish species Hoplias intermedius. Toxicology Reports 5:1032-1043.
- Zhang MZ, Li M, Wang RX, Qian YX 2018. Effects of acute ammonia toxicity on oxidative stress, immune response and apoptosis of juvenile yellow catfish Pelteobagrus fulvidraco and the mitigation of exogenous taurine. Fish Shellfish Immun 79:313-320.