A Protective Role of Coenzyme Q10 in Ethambutol-Induced Retinal Ganglion Cell Toxicity: A Randomised Controlled Trial in Mice

ABSTRACT

Ethambutol is a widely used drug to treat tuberculosis that may cause visual disturbance including ethambutol toxic optic neuropathy (ETON). The disease disrupts bodily tissues’ energy production, including the retinal ganglion cells (RGC). Many have proposed treatment with coenzyme Q10 (coQ10) due to its antioxidant and facilitative effects that can improve mitochondrial electron transport. The present study hence assessed whether coQ10 could protect against ETON through a parallel triple-blinded randomised controlled trial in 18 mice using computer-generated tables for treatment allocation. All of the mice received 25 mg/kg ethambutol daily, while only nine in the treated group also received 100 mg/kg coQ10. After 30 days, blinded pathologists counted RGC numbers in enucleated and dyed orbital tissue. The treated group had significantly denser RGCs at 47.2 (standard deviation [SD] 10.6) cells per 500 µm microscope field vs 33.5 (SD 6.3) in the control group (t = 3.34, p = .004). CoQ10 therefore protected RGCs from ETON. Clinical trials of coQ10 in human subjects treated with ethambutol should be considered.

Introduction

Tuberculosis (TB) is one of the most prevalent infectious diseases, especially in developing or low-income countries. Mycobacterium tuberculosis (MTB) infects almost a quarter (~23%) of the world’s population and causes 1,500,000 deaths per year.¹ The disease ranks at the top for transmission due to its airborne transmission and the occurrence of many unreported cases (> 29%).¹

Indonesia ranks second on the “high burden country” list of TB.² Within the fourth most populated archipelago, TB and other respiratory infections have been listed as the fifth highest cause of death after cardiovascular disease, neoplasms, diabetes, kidney diseases, and digestive diseases.³ There were 845,000 confirmed cases and 13,947 deaths due to TB in Sumatra and Java in 2020.⁴ Standard treatment of TB involves a 6 month regimen of rifampicin, isoniazid, pyrazinamide, and ethambutol. Nonetheless, each of the medications has its own particular adverse effects: skin rash due to isoniazid; purpura due to rifampicin; arthralgia due to pyrazinamide; and orbital toxicity due to ethambutol.⁵

Ethambutol toxic optic neuropathy (ETON) is one of the major effects of excessive or prolonged ethambutol use, leading to 100,000 cases of blindness annually.⁶ The toxicity often occurs on days 39–40 of treatment, however toxic effects may appear as soon as 2 weeks of treatment.⁷ There is a dose-dependent relationship with ETON occurring in < 1% of cases on 15 mg/kg daily ethambutol, 5–6% of cases on 25 mg/kg, and 18% of cases on over 35 mg/kg.⁸ Structures with higher local accumulation of ethambutol have a higher risk of primary damage. Retinal ganglionic cells (RGC) have a high affinity for ethambutol therefore explaining the high incidence of ETON.^9,10

Zinc, coenzyme Q10 (coQ10), and Buqihuoxue formula with methycobal combination have been touted as possible treatments for ETON.^11,12 CoQ10 or ubiquinone is a lipid-soluble molecule on cell membranes and in mitochondria. The substance occurs naturally through the biological processes of cellular metabolism and respiration, or can come from foods and beverages (e.g., meats, fish, nuts, oils, and dairy produce).^13,14 Besides its antioxidant and organ protection abilities, coQ10 plays an integral role in cellular metabolism and oxidative management by supporting energy biosynthesis in mitochondria.¹⁵ These effects could be beneficial in the prevention and treatment of ETON.

Previously, the effects of coQ10 on contrast sensitivity in ETON,¹⁶ and on RGC disruption in experimental methanol toxicity in rats have been studied.¹⁷ In this study we are evaluating the ability of coQ10 to prevent damage to RGCs in mice being treated with ethambutol.

Materials & methods

Study design

The experimental study was conducted in Padjadjaran University, Hasan Sadikin Hospital, and Cicendo Eye Hospital from June to July 2014. The randomised, parallel, and triple-blinded study followed the Helsinki Declaration and institutional review board standards through the Ethics Committee of Hasan Sadikin General Hospital certification of LB.04.01/A05/EC/278/VI/2014. Furthermore, the study adheres to the Association for Research in Vision and Ophthalmology statement for animal use in ophthalmic and vision research. The authors computed a minimal sample size of 18 subjects via 5% alpha and 90% power.

Subject enrolment, handling, and care

The Pharmacology clinic of Padjadjaran University supplied the mice and instruments for the study. All 18 mice were controlled in terms of gender, age, body weight, and health. They were 10-week-old male mice weighing 300 grams without any illnesses, anatomical issues, or functional anomalies of the eyes. The mice had been acclimatised to the Padjadjaran laboratory conditions for 21 days before the study in a single large cage with 20–22^OC temperature and 40–60% humidity. They were then distributed equally to two separate clusters via random computer-generated tables. Mice were excluded if they lost ≥ 10% of their initial weight, died, or procured inadequate histopathology samples at any point of the study.

National and institutional animal standardised handling protocols were done within all study phases to reduce any pain or discomfort. Animal caretakers allowed unlimited consumption of pathogen-free food and clean water. Anaesthesia for the enucleation surgery used a combination of isoflurane 3.0% and 0.5 L/min of oxygen.

Materials & instruments

The present study used various selected and tested equipment. Examples of the most important materials are: (1) ethambutol hydrochloride; (2) coQ10; (3) 10% concentrated formalin; (4) light microscope; (5) enucleation surgery tools; and (6) histology examination tools. Table 1 shows a complete list of the materials needed.

Table 1.

Complete list of equipment.

Materials & Tools
Ethambutol hydrochloride	Coenzyme Q10	Digital scale	Spatula
Distilled water	Ketamine	Surgery table	Pipette
Formalin (10% & buffer)	Alcohol (70–90%)	1- & 3-ml syringe	Light microscope
Xylol	Haematoxylin	Glass object slide	Cover glass
Eosin	Paraffin	Enucleation equipment

Blinding technique

Biases can affect a study’s integrity. They may render any results invalid or vague. The current study responded to the problem through a triple masking methodology, where all participants, interventionists, and result assessors were blinded.

We easily achieved the first criteria of ensuring the protocols and medicine anonymity from the subjects since this was an animal study with mice. Meanwhile, the usage of single letter labelled medicine tubes blinded the interventionists (i.e., an outsourced pharmacist switched the coQ10 and ethambutol labels and with labels marked “A” and “B”). Two other measures were taken to conceal the group origin of the mice. The interventionists guarded the mice room in shifts and prohibited any surgical personnel, pathology team, and unauthorised person from entering the room with the mice before the last day of the intervention. Furthermore, mice were only labelled as coming from group one or two during the enucleation surgery and the pathology assessment. The study also employed independent and outsourced data curators and analysts to deal with the result and statistics.

Data collection and measurement

This study randomly divided the sample into the treated and control groups with nine mice each. For 30 days, interventionists gave daily 25 mg/kg body weight (BW) ethambutol hydrochloride and 100 mg/kg BW coQ10 to the treated group mice, whereas the control group mice only received the daily ethambutol hydrochloride. The 30 day intervention period was deemed appropriate for the dose of ethambutol used since Heng et al. found that ethambutol-induced RGC toxicity could be detected by as early as 1 week.¹⁰

All mice underwent orbital enucleation surgery on the morning of day 31. Peri-operative tissue samples were then preserved in formalin and adapted to the paraffin block. Pathologists afterwards treated the samples with haematoxylin and eosin (H&E) dyes to aid further inspection. Figures 1 and 2 show the specific instructions on tissue handling.^18,19 The pathologists subsequently counted the RGCs per 500 μm field using a light microscope with 400 x magnification.

Figure 1.

Tissue and paraffin preparation.

Figure 2.

Haematoxylin and eosin staining techniques.

Statistical analysis

Analysts tabulated the data in Microsoft Excel 365 (Microsoft, Redmond, Washington, USA) and then performed statistical analysis in Statistical Product and Service Solutions 26 (SPSS, Armonk, New York, USA) using an independent t-test to measure the effect of coenzyme Q10 on RGC numbers. P-values less than .05 marked any significance.

Results

This 1 month experimental study of the mice upheld a 100% participation rate, with no mice dying and none losing ≥ 10% of their body weight during the study. Table 2 shows the RGC counts in the mice that did or did not receive CoQ10.

Table 2.

Retinal ganglion cell numbers per 500 μm field in mice that did or did not receive coenzyme Q10 supplementation.

	Total	Coenzyme Q10 Supplementation		t (Effect size)	p
		Treated (n = 9)	Untreated (n = 9)
Mean (SD)	40.4 (11.0)	47.2 (10.6)	33.5 (6.3)	3.34 (1.58)	.004
Median	38.5	46.3	30.3
Range	27.7–62.3	33.0–62.3	27.7–45.0

 SD = standard deviation

Mice from the treated group had a mean 47.2 (standard deviation [SD] 10.6) RGCs per 500 μm microscope field compared with 33.5 (SD 6.3) RGCs in the control mice (t = 3.34, p = .004, with a 1.58 Cohen’s d effect size). H&E dyed microscopic observation between groups also portrayed a similar morphologic trend. The mice who had received coenzyme Q10 supplementation had RGCs with a more coherent structure (Figure 3a) compared with the mice that had not (Figure 3b).

Figure 3.

Haematoxylin and eosin stained histological Appearance (400 x) of retinal ganglion cells in ethambutol treated mice with (a) and without (b) coenzyme Q10 supplementation.

Discussion

Ethambutol is a necessary treatment for TB. It enters the bacteria and acts as bacteriostatic agent by inhibiting arabinosyltransferase synthesis and function.^20–22 Accumulated mycolic acid and trehalose isomers from this inhibition interfere with MTB cell wall and cell division and tag the bacteria for destruction by immune cells.^20,23

ETON strongly correlates with the accumulation of lysosomes despite its uncertain pathophysiology. It has been hypothesised that ethambutol causes lysosomal neutralisation and autophagy impairment by gathering excessive zinc within the lysosome.²⁴ It destroys the RGC through the generation of a toxin with a similar nature to glutamate-mediated toxicity. Overstimulation of N-methyl-D-aspartate receptors by excess glutamate causes massive calcium ion intake which leads to RGC destruction.¹⁰

The calcium imbalance that occurs between mitochondria and the cytosol suggests that ethambutol toxicity is due to its effects on mitochondrial respiration and the resulting flow of calcium from cytosolic pools into the mitochondria. This coupled with its metal chelating ability enables ethambutol to interfere with mitochondrial function and create numerous reactive oxygen species.^25,26

We used an ethambutol dose of 25 mg/kg as this is the lowest dose that can still trigger ETON. The World Health Organisation has reported that ocular toxicity became apparent from 25 mg/kg upwards compared with lesser dosages (0–16% versus 0–13%).²⁷ Koul in his review further commented that the incidence of ocular toxicity was 1% with a dose of 15 mg/kg of ethambutol compared with 5–6% on 25 mg/kg.⁸

Many therapies like zinc, coQ10, and Buqihuoxue formula with methycobal combination have the potentials to circumvent this toxicity.^11,12 coQ10 is a lipid-soluble substance comprised of 59 carbon, 90 hydrogen, and four oxygen atoms (863.3 gram/mol).²⁸ We chose a 100 mg/kg BW daily coQ10 dose following earlier research, where a decrease in reactive oxygen species was more apparent at 100 mg/kg dose compared with 25 or 50 mg/kg.²⁹

CoQ10 assists in generating adenosine triphosphate through the electron transport chain inside mitochondria. It notably helps move electrons between complex I–III by temporarily accepting them from succinate or reduced nicotinamide adenine dinucleotide.³⁰ It also serves in the cell membrane as a potent defence against oxidative damage by reactive oxygen species by stimulating powerful antioxidant secretion (e.g., superoxide dismutase).^31,32

Several studies have shown that coQ10 protects against cardiovascular, fertility, renal, neurodegenerative, and metabolic diseases.¹³ A 2017 study established coQ10 as a protective measure against methanol’s toxicity to RGCs (∆ = 1.79, p < .001).¹⁷ Our results have shown a protective effect against ethambutol toxicity to RGCs. An effect size of 1.58 shows that the coQ10 protection may be applicable to a large population, ensuring over 92% expected mean difference.

The limitations of this study are its small sample size, the lack of establishing the optimum dosage of coQ10, and no measurement of any other factors that may influence ethambutol toxicity on RGCs. We tried to mitigate the last factor by ensuring the health of the mice circumventing possibilities of the RGCs being affected by any physical and chemical trauma.

Conclusion

We have provided evidence that coQ10 is protective against ethambutol-induced toxicity to RGCs in mice. Clinical trials should be considered for coQ10 supplementation in humans treated with ethambutol and in other conditions where there is damage to mitochondria in RGCs. In addition, there should be more study on its pharmacological and pharmacokinetic properties.

Funding Statement

The author(s) reported there is no funding associated with the work featured in this article.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability

Data may be requested to the corresponding author upon reasonable reasons