Application of LDH-Release Assay to Cellular-Level Evaluation of the Toxic Potential of Harmful Algal Species

Zou, Yu; Kim, Daekyung; Yagi, Minoru; Yamasaki, Yasuhiro; Kurita, Jun; Iida, Takaji; Matsuyama, Yukihiko; Yamaguchi, Kensei; Oda, Tatsuya

doi:10.1271/bbb.120764

Cited by 20 publications

(14 citation statements)

References 36 publications

(44 reference statements)

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“…LDH release has been demonstrated to be a reliable biochemical indicator of cell damage in previous studies [46]–[48]. In agreement with confocal microscopy results, HMEECs infected with WT P. aeruginosa demonstrated minimal LDH release by 8h post-infection at an MOI of 1 and 10 (Figure 9 and Figure S3).…”

Section: Resultssupporting

confidence: 88%

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In Vitro Interaction of Pseudomonas aeruginosa with Human Middle Ear Epithelial Cells

et al. 2014

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BackgroundOtitis media (OM) is an inflammation of the middle ear which can be acute or chronic. Acute OM is caused by Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis whereas Pseudomonas aeruginosa is a leading cause of chronic suppurative otitis media (CSOM). CSOM is a chronic inflammatory disorder of the middle ear characterized by infection and discharge. The survivors often suffer from hearing loss and neurological sequelae. However, no information is available regarding the interaction of P. aeruginosa with human middle ear epithelial cells (HMEECs).Methodology and FindingsIn the present investigation, we demonstrate that P. aeruginosa is able to enter and survive inside HMEECs via an uptake mechanism that is dependent on microtubule and actin microfilaments. The actin microfilament disrupting agent as well as microtubule inhibitors exhibited significant decrease in invasion of HMEECs by P. aeruginosa. Confocal microscopy demonstrated F-actin condensation associated with bacterial entry. This recruitment of F-actin was transient and returned to normal distribution after bacterial internalization. Scanning electron microscopy demonstrated the presence of bacteria on the surface of HMEECs, and transmission electron microscopy confirmed the internalization of P. aeruginosa located in the plasma membrane-bound vacuoles. We observed a significant decrease in cell invasion of OprF mutant compared to the wild-type strain. P. aeruginosa induced cytotoxicity, as demonstrated by the determination of lactate dehydrogenase levels in culture supernatants of infected HMEECs and by a fluorescent dye-based assay. Interestingly, OprF mutant showed little cell damage compared to wild-type P. aeruginosa.Conclusions and SignificanceThis study deciphered the key events in the interaction of P. aeruginosa with HMEECs in vitro and highlighted the role of bacterial outer membrane protein, OprF, in this process. Understanding the molecular mechanisms in the pathogenesis of CSOM will help in identifying novel targets to design effective therapeutic strategies and to prevent hearing loss.

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Section: Resultssupporting

confidence: 88%

“…LDH is a stable cytosolic enzyme and has been used as an indicator of cell damage [46]–[48]. HMEECs were infected with bacteria at various MOI and for different time periods.…”

Section: Methodsmentioning

confidence: 99%

In Vitro Interaction of Pseudomonas aeruginosa with Human Middle Ear Epithelial Cells

et al. 2014

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“…LDH activity in the culture medium was measured by a commercial LDH assay kit (Roche, Mannheim, Germany) according to the supplier’s manual [22]. Absorbance of the samples was detected at 490 nm on a spectrophotometer (Victor3, Perkin Elmer, Waltham, MA, USA).…”

Section: Methodsmentioning

confidence: 99%

Adiponectin Relieves Human Adult Cardiac Myocytes Injury Induced by Intermittent Hypoxia

Zhang

Rui

et al. 2019

Med Sci Monit

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BackgroundObstructive sleep apnea (OSA) is associated with many cardiovascular disorders. Intermittent hypoxia (IH) is a key pathological hallmark of OSA. This study was conducted to evaluate the potential therapeutic effects and the associated mechanisms of adiponectin (APN) on IH induced human adult cardiac myocytes (HACMs) injury.Material/MethodsHACMs were exposed to normoxia or IH (1% to 21% O2) using a novel cell culture bio-reactor with gas-permeable membranes. Cell viability was detected by Cell Counting Kit-8 assay. Cell membrane integrity was assessed by the detection of lactate dehydrogenase (LDH) release. Cell apoptosis was analyzed by flow cytometry. Malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) levels were determined using specific assay kits. P-AMPK (AMP-activated protein kinase), p-LKB1, and p-p65 protein levels were measured by western blotting. Pro-inflammatory factors including interleukin (IL)-1β, IL-6, IL-8 expressions were detected by enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction.ResultsThe results showed that APN had no cytotoxic to HACMs. Compared with the control group, HACMs cell viability significantly decreased, LDH release increased and cell apoptosis increased in the IH group. The levels of IL-1β, IL-6, IL-8, MDA, and p-p65 were higher, while the levels of SOD, GSH-Px, p-AMPK, and p-LKB1 were lower in HACMs cells in the IH group than that in the control group. However, APN treatment significantly rescued these effects compared with the IH group in a dose-dependent manner.ConclusionsIn conclusion, these results indicated that APN protected against IH induced HACMs injury possibly mediated by AMPK and NF-κB pathway.

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“…Once the 2-hr exposure was completed, algal cultures in the inserts were mixed and 1.5 mL aliquots transferred into Eppendorf tubes, and centrifuged at 370 ×g for 5 min. Supernatant was recovered and stored at -20°C until analysis of the enzyme lactate dehydrogenase (LDH) was performed to determine whether damage to cellular membrane occurred in association with changes in gill cell viability, as LDH activity is usually associated with cytotoxicity [ 38 ].…”

Section: Methodsmentioning

confidence: 99%

“…The assay used for the quantification of this enzyme was reproduced from Zou et al [ 38 ]. LDH-detection assay mix was prepared with 543 μM nitro blue tetrazolium (N5514, Sigma), 814 μM β-NAD + (N7004, Sigma), 20 U mL -1 diaphorase (D2197, Sigma) and 50 mM lactic acid sodium salt (BIOLB0571, Astral Scientific) in 10 mM Tris-HCl buffer at pH 8.5.…”

Section: Methodsmentioning

confidence: 99%

Progress in Understanding Algal Bloom-Mediated Fish Kills: The Role of Superoxide Radicals, Phycotoxins and Fatty Acids

et al. 2015

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Quantification of the role of reactive oxygen species, phycotoxins and fatty acids in fish toxicity by harmful marine microalgae remains inconclusive. An in vitro fish gill (from rainbow trout Oncorhynchus mykiss) assay was used to simultaneously assess the effect in superoxide dismutase, catalase and lactate dehydrogenase enzymatic activities caused by seven species of ichthyotoxic microalgae (Chattonella marina, Fibrocapsa japonica, Heterosigma akashiwo, Karenia mikimotoi, Alexandrium catenella, Karlodinium veneficum, Prymnesium parvum). Quantification of superoxide production by these algae was also performed. The effect of purified phycotoxins and crude extracts was compared, and the effect of fatty acids is discussed. The raphidophyte Chattonella was the most ichthyotoxic (gill cell viability down to 35%) and also the major producer of superoxide radicals (14 pmol cell-1 hr-1) especially after cell lysis. The raphidophyte Heterosigma and dinoflagellate Alexandrium were the least toxic and had low superoxide production, except when A. catenella was lysed (5.6 pmol cell-1 hr-1). Catalase showed no changes in activity in all the treatments. Superoxide dismutase (SOD) and lactate dehydrogenase exhibited significant activity increases of ≤23% and 51.2% TCC (total cellular content), respectively, after exposure to C. marina, but SOD showed insignificant changes with remaining algal species. A strong relationship between gill cell viability and superoxide production or superoxide dismutase was not observed. Purified brevetoxins PbTx-2 and -3 (from Karenia brevis, LC50 of 22.1 versus 35.2 μg mL-1) and karlotoxin KmTx-2 (from Karlodinium; LC50 = 380 ng mL-1) could almost entirely account for the fish killing activity by those two dinoflagellates. However, the paralytic shellfish toxins (PST) GTX1&4, C1&C2, and STX did not account for Alexandrium ichthyotoxicity. Only aqueous extracts of Alexandrium were cytotoxic (≤65% decrease of viability), whereas crude methanol and acetone extracts of Chattonella, Fibrocapsa, Heterosigma, Karlodinium and Prymnesium decreased cell viability down to 0%. These and our previous findings involving the role of fatty acids confirm that superoxide radicals are only partially involved in ichthyotoxicity and point to a highly variable contribution by other compounds such as lipid peroxidation products (e.g. aldehydes).

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Application of LDH-Release Assay to Cellular-Level Evaluation of the Toxic Potential of Harmful Algal Species

Cited by 20 publications

References 36 publications

In Vitro Interaction of Pseudomonas aeruginosa with Human Middle Ear Epithelial Cells

In Vitro Interaction of Pseudomonas aeruginosa with Human Middle Ear Epithelial Cells

Adiponectin Relieves Human Adult Cardiac Myocytes Injury Induced by Intermittent Hypoxia

Progress in Understanding Algal Bloom-Mediated Fish Kills: The Role of Superoxide Radicals, Phycotoxins and Fatty Acids

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