Standardization of a new method for assessing the development of cataract in cultured bovine lenses

Segewkal Heruye, Leonce N. Maffofou N., Neetu U. Singh, Dan Munt, Ya Fatou Njie-Mbye, Sunny E. Ohia, Catherine A. Opere

Research output: Contribution to journalArticle

Abstract

Purpose: To standardize a new method for assessing cataractogenesis in isolated cultured bovine lenses using L-cysteine as the standard anti-cataract agent. Methods: Intact bovine lenses were cultured in DMEM with L-cysteine in presence or absence of hydrogen peroxide (H2O2). Lens opacity (transmittance) was determined using a plate reader. Lens homogenate glutathione (GSH) and superoxide dismutase (SOD) contents were measured using enzyme immunoassays kits. Results: DMEM-cultured lenses exhibited a time-dependent loss in transmittance (230–710 nm) up to 120 h, achieving the highest reduction of 38.6 ± 0.09% at 420 nm (p < .001;n = 12). Compared to untreated lenses (time in hours [t] = 0), L-cysteine (10−6 M and 10−5 M) significantly (p < .001;n = 6) increased time-dependent transmittance (420 nm) by 31.6 ± 0.17% and 28.0 ± 0.07%(t = 120), respectively. When compared to DMEM-cultured lenses (t = 0), H2O2 (10 mM, 50 mM and 100 mM) significantly (p < .001;n = 12) reduced transmittance by 57.8 ± 0.1, 57.4 ± 0.04 and 87.7 ± 0.6%(t = 120), respectively. Moreover, L-cysteine significantly (p < .001;n = 6) attenuated H2O2 (50 mM)-induced decrease in transmittance by 12.5 ± 0.05%(10−6 M), 13.0 ± 0.09%(10−5 M), 14.5 ± 0.08%(10−4 M) and 8.6 ± 0.11%(10−3 M)(t = 120), respectively. When compared to untreated lenses (t = 0), the time-dependent decrease (p < .001;n = 5) in lenticular total GSH content and total SOD activity of 46.1 ± 0.06% and 42.0 ± 1.65% (t = 120) was attenuated (p < .001;n = 5) by L-cysteine (10−6 M) by 76.6 ± 0.06% and 7.4 ± 1.98%, respectively. Similarly, the H2O2(50 mM)-induced decline (p < .001; n = 5) in total GSH content and SOD activity of 82.6 ± 0.08% and 86.6 ± 0.66% (t = 120) was attenuated by L-cysteine (10−4 M) by 74.7 ± 1.05% and 161.1 ± 4.9%, respectively. Conclusion: Measurement of spectral transmission coupled with assessment of the activity of antioxidant enzymes in bovine cultured lens can provide a useful tool in studies of cataracts in an animal model of this disease.

Original languageEnglish (US)
Article number106592
JournalJournal of Pharmacological and Toxicological Methods
Volume98
DOIs
StatePublished - Jul 1 2019

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Cataract
Standardization
Lenses
Cysteine
Superoxide Dismutase
Animal Disease Models
Immunoenzyme Techniques
Opacity
Enzymes
Hydrogen Peroxide
Glutathione
Antioxidants
Animals

All Science Journal Classification (ASJC) codes

  • Toxicology
  • Pharmacology

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Standardization of a new method for assessing the development of cataract in cultured bovine lenses. / Heruye, Segewkal; Maffofou N., Leonce N.; Singh, Neetu U.; Munt, Dan; Njie-Mbye, Ya Fatou; Ohia, Sunny E.; Opere, Catherine A.

In: Journal of Pharmacological and Toxicological Methods, Vol. 98, 106592, 01.07.2019.

Research output: Contribution to journalArticle

Heruye, Segewkal ; Maffofou N., Leonce N. ; Singh, Neetu U. ; Munt, Dan ; Njie-Mbye, Ya Fatou ; Ohia, Sunny E. ; Opere, Catherine A. / Standardization of a new method for assessing the development of cataract in cultured bovine lenses. In: Journal of Pharmacological and Toxicological Methods. 2019 ; Vol. 98.
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title = "Standardization of a new method for assessing the development of cataract in cultured bovine lenses",
abstract = "Purpose: To standardize a new method for assessing cataractogenesis in isolated cultured bovine lenses using L-cysteine as the standard anti-cataract agent. Methods: Intact bovine lenses were cultured in DMEM with L-cysteine in presence or absence of hydrogen peroxide (H2O2). Lens opacity (transmittance) was determined using a plate reader. Lens homogenate glutathione (GSH) and superoxide dismutase (SOD) contents were measured using enzyme immunoassays kits. Results: DMEM-cultured lenses exhibited a time-dependent loss in transmittance (230–710 nm) up to 120 h, achieving the highest reduction of 38.6 ± 0.09{\%} at 420 nm (p < .001;n = 12). Compared to untreated lenses (time in hours [t] = 0), L-cysteine (10−6 M and 10−5 M) significantly (p < .001;n = 6) increased time-dependent transmittance (420 nm) by 31.6 ± 0.17{\%} and 28.0 ± 0.07{\%}(t = 120), respectively. When compared to DMEM-cultured lenses (t = 0), H2O2 (10 mM, 50 mM and 100 mM) significantly (p < .001;n = 12) reduced transmittance by 57.8 ± 0.1, 57.4 ± 0.04 and 87.7 ± 0.6{\%}(t = 120), respectively. Moreover, L-cysteine significantly (p < .001;n = 6) attenuated H2O2 (50 mM)-induced decrease in transmittance by 12.5 ± 0.05{\%}(10−6 M), 13.0 ± 0.09{\%}(10−5 M), 14.5 ± 0.08{\%}(10−4 M) and 8.6 ± 0.11{\%}(10−3 M)(t = 120), respectively. When compared to untreated lenses (t = 0), the time-dependent decrease (p < .001;n = 5) in lenticular total GSH content and total SOD activity of 46.1 ± 0.06{\%} and 42.0 ± 1.65{\%} (t = 120) was attenuated (p < .001;n = 5) by L-cysteine (10−6 M) by 76.6 ± 0.06{\%} and 7.4 ± 1.98{\%}, respectively. Similarly, the H2O2(50 mM)-induced decline (p < .001; n = 5) in total GSH content and SOD activity of 82.6 ± 0.08{\%} and 86.6 ± 0.66{\%} (t = 120) was attenuated by L-cysteine (10−4 M) by 74.7 ± 1.05{\%} and 161.1 ± 4.9{\%}, respectively. Conclusion: Measurement of spectral transmission coupled with assessment of the activity of antioxidant enzymes in bovine cultured lens can provide a useful tool in studies of cataracts in an animal model of this disease.",
author = "Segewkal Heruye and {Maffofou N.}, {Leonce N.} and Singh, {Neetu U.} and Dan Munt and Njie-Mbye, {Ya Fatou} and Ohia, {Sunny E.} and Opere, {Catherine A.}",
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T1 - Standardization of a new method for assessing the development of cataract in cultured bovine lenses

AU - Heruye, Segewkal

AU - Maffofou N., Leonce N.

AU - Singh, Neetu U.

AU - Munt, Dan

AU - Njie-Mbye, Ya Fatou

AU - Ohia, Sunny E.

AU - Opere, Catherine A.

PY - 2019/7/1

Y1 - 2019/7/1

N2 - Purpose: To standardize a new method for assessing cataractogenesis in isolated cultured bovine lenses using L-cysteine as the standard anti-cataract agent. Methods: Intact bovine lenses were cultured in DMEM with L-cysteine in presence or absence of hydrogen peroxide (H2O2). Lens opacity (transmittance) was determined using a plate reader. Lens homogenate glutathione (GSH) and superoxide dismutase (SOD) contents were measured using enzyme immunoassays kits. Results: DMEM-cultured lenses exhibited a time-dependent loss in transmittance (230–710 nm) up to 120 h, achieving the highest reduction of 38.6 ± 0.09% at 420 nm (p < .001;n = 12). Compared to untreated lenses (time in hours [t] = 0), L-cysteine (10−6 M and 10−5 M) significantly (p < .001;n = 6) increased time-dependent transmittance (420 nm) by 31.6 ± 0.17% and 28.0 ± 0.07%(t = 120), respectively. When compared to DMEM-cultured lenses (t = 0), H2O2 (10 mM, 50 mM and 100 mM) significantly (p < .001;n = 12) reduced transmittance by 57.8 ± 0.1, 57.4 ± 0.04 and 87.7 ± 0.6%(t = 120), respectively. Moreover, L-cysteine significantly (p < .001;n = 6) attenuated H2O2 (50 mM)-induced decrease in transmittance by 12.5 ± 0.05%(10−6 M), 13.0 ± 0.09%(10−5 M), 14.5 ± 0.08%(10−4 M) and 8.6 ± 0.11%(10−3 M)(t = 120), respectively. When compared to untreated lenses (t = 0), the time-dependent decrease (p < .001;n = 5) in lenticular total GSH content and total SOD activity of 46.1 ± 0.06% and 42.0 ± 1.65% (t = 120) was attenuated (p < .001;n = 5) by L-cysteine (10−6 M) by 76.6 ± 0.06% and 7.4 ± 1.98%, respectively. Similarly, the H2O2(50 mM)-induced decline (p < .001; n = 5) in total GSH content and SOD activity of 82.6 ± 0.08% and 86.6 ± 0.66% (t = 120) was attenuated by L-cysteine (10−4 M) by 74.7 ± 1.05% and 161.1 ± 4.9%, respectively. Conclusion: Measurement of spectral transmission coupled with assessment of the activity of antioxidant enzymes in bovine cultured lens can provide a useful tool in studies of cataracts in an animal model of this disease.

AB - Purpose: To standardize a new method for assessing cataractogenesis in isolated cultured bovine lenses using L-cysteine as the standard anti-cataract agent. Methods: Intact bovine lenses were cultured in DMEM with L-cysteine in presence or absence of hydrogen peroxide (H2O2). Lens opacity (transmittance) was determined using a plate reader. Lens homogenate glutathione (GSH) and superoxide dismutase (SOD) contents were measured using enzyme immunoassays kits. Results: DMEM-cultured lenses exhibited a time-dependent loss in transmittance (230–710 nm) up to 120 h, achieving the highest reduction of 38.6 ± 0.09% at 420 nm (p < .001;n = 12). Compared to untreated lenses (time in hours [t] = 0), L-cysteine (10−6 M and 10−5 M) significantly (p < .001;n = 6) increased time-dependent transmittance (420 nm) by 31.6 ± 0.17% and 28.0 ± 0.07%(t = 120), respectively. When compared to DMEM-cultured lenses (t = 0), H2O2 (10 mM, 50 mM and 100 mM) significantly (p < .001;n = 12) reduced transmittance by 57.8 ± 0.1, 57.4 ± 0.04 and 87.7 ± 0.6%(t = 120), respectively. Moreover, L-cysteine significantly (p < .001;n = 6) attenuated H2O2 (50 mM)-induced decrease in transmittance by 12.5 ± 0.05%(10−6 M), 13.0 ± 0.09%(10−5 M), 14.5 ± 0.08%(10−4 M) and 8.6 ± 0.11%(10−3 M)(t = 120), respectively. When compared to untreated lenses (t = 0), the time-dependent decrease (p < .001;n = 5) in lenticular total GSH content and total SOD activity of 46.1 ± 0.06% and 42.0 ± 1.65% (t = 120) was attenuated (p < .001;n = 5) by L-cysteine (10−6 M) by 76.6 ± 0.06% and 7.4 ± 1.98%, respectively. Similarly, the H2O2(50 mM)-induced decline (p < .001; n = 5) in total GSH content and SOD activity of 82.6 ± 0.08% and 86.6 ± 0.66% (t = 120) was attenuated by L-cysteine (10−4 M) by 74.7 ± 1.05% and 161.1 ± 4.9%, respectively. Conclusion: Measurement of spectral transmission coupled with assessment of the activity of antioxidant enzymes in bovine cultured lens can provide a useful tool in studies of cataracts in an animal model of this disease.

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