alpha lipoic acid and Nrf2

This post was inspired by a health care guru of sorts who harped upon the wonders of NRF2 and all of the genes it controls the transcription of. The transcription factor Nrf2 and its redox sensitive keeper Keap1 have been covered in a post on ozone therapy. This post points to Nrf2 as the mediator of α-LA protection against As, Hg, and Cd toxicity and GSH synthesis as an important part of the protection.

Arsenic and α–lipoic acid in HepG2 cells [1]

This study out of Guadalajara Mexico. The authors used the hepatocyte cell line Hep2G. The idea was to test the hypothesis that alpha lipoic acid could increase Nrf2 transcription factor gene transcripts. In this case the 8 hour pre treatment was 5 mM α-lipoic acid and the challenge 50μM As³⁺.

1. The MTT assay is a color changing assay that relies on mitochondria being functional. Glutathione (GSH is a reducing agent that helps maintain redox balance in the cell. 5 mM α-lipoic acid increased MTT activity by perhaps 10% over control and 50μM As³⁺, which was not toxic at this level. GSH levels were unchanged.
2. When 5 mM α-lipoic was given prior to the MTT activity (cell viability) and GSH levels in response to 50μM As³⁺ were not statistically different.
3. These treatments alone had no influence on ACu/Zn super oxide dismutase 1, glutathione S-transferase 1, or apoptosis protease caspase 3 transcripts. Hemoxygenase 1 transcripts increased over 25x with just 50μM As³⁺. A modest increase was seen in metallothionene transcripts. Both treatments increased GCLM but not GCLC transcripts. A Western blot was presented showing that both treatments increased Nrf2 levels.
4. The 8 hour 5 mM α-lipoic acid pretreatment blunted some of the very dramatic 50μM As³⁺induced increase in hemoxygenase 1 transcripts.

The porphyrin of heme minus the iron and other modifications gives us biliverdin.

Cadmium and α–lipoic acid in HepG2 cells, GSH synthesis [2]

The same group from Guadalajara also examined the interplay between

Gene	ID	Control	5 mM ALA	1 μM Cd2+	+5 μM Cd2+	5 mM ALA +5 μM Cd2+	1 μM Cd2+ +5 μM Cd2+	*
Glutamylcysteine synthetase catalytic subunit	GCLC	1	4.1±0.4+	1.5±0.1+	7.6±0.3+	3.9±0.3++	5++
Glutamyl cysteine synthetase modulatory subunit	GCLM	1	2.4+	1.4	19+	9.5	12.++
Metallothionein 1A	MT1A	1	1.3	2.2+	13+	9++	10
Heme oxygenase 1	HMOX1	1	0.8+	2.0+	52.6+	34++	45++
Interleukin-6	IL-6	1	1.6	2+	19+	13++	15.++
Tumor necrosis factor alpha	TNF	1	1.5	1.6+	9+	5.++	8++

Cadmium and α–lipoic acid in HepG2 cells, Part 1 [3]

This Chinese study by Shi and coauthors was similar to the Mexican study in some ways. HepG2 cells were treated with 100μM α-LA and 25μM cadmium chloride both for 16h respectively. In the co-treatment groups, HepG2 cells were pre-treated with α-LA
(10, 50 and 100μM) for 8h, followed by treatment with 25μM cadmium for 16h in the
presence of α-LA

1. Shi and coauthors also looked at cell viability with the expected decrease with cadmium, a slight boost with 100μM α-LA, and a mitigation of the cadmium toxicity with ramping does of 100μM α-LA.
2. Reactive oxygen species generation was measured by the oxidation of dichlorofluorescein to a fluorescent compound. 100μM α-LA was without an effect while 25μM cadmium increased generation of ROS that was mitigated by increasing doses of α-LA.
3. 100μM α-LA slightly bumped up the concentration of reduced glutathione. Reduced GSH was cut in half by 25μM cadmium. The 10, 50 and 100μM α-LA ramp mitigated this effect of cadmium. The authors also measured the ratio of reduced to oxidized glutathione, which mirrored this trend.
4. Glutathione reductase enzyme protein levels, mRNA transcript levels, and activity were measured. The protein levels were more effected by cadmium than per molecule activity or mRNA levels. Ramping up α-LA mitigated the effect of cadmium.
5. Nrf2 phosphorylation (activation) was reduced by 25μM cadmium but brought to above control levels by ramping up α-LA.
6. Some not so clear images of Nrf-2 nuclear translocation were shown.

Cadmium and α–lipoic acid in HepG2 cells, part 2 [4]

HepG2 cells were treated with 50 μM α-LA, 25 μM cadmium for 16 h, respectively. For α-LA and cadmium co-treatment, the cells were pretreated with 50 μM -LA for 8 h, followed by treatment with 25 M cadmium for 16 h in the presence of α-LA. For α-LA + cadmium + brusatol co-treatment, the cells were pretreated with 50 μM -αLA and brusatol for 8 h, followed by exposure to 25 μM cadmium for 16 h in the presence of α-LA and plus or minus the Nrf2 blocker brusatol.

summary of figures, findings

Since the Zhang study was not published in a public access journal, only findings outlined in the figures will be presented.

1. 50 μM α-LA increased protein level of Nrf2 and the amount of which was phosphorylated (nucleus targeted). 25 μM cadmium had the opposite effect. α-LA mitigated the cadmium effect. Brusatol reversed the α-LA improvement.
2. Cadmium was a strong inducer of both the regulatory and catalytic subunits of glutamyl cysteine ligase. α-LA in combination increased transcripts more. Brusatol inhibited this increase.
3. α-LA increased protein levels and mRNA transcripts of glutathione reuductase. Cadmium increased both. α-LA, in combination with cadmium, mitigated this decrease. This effect was blocked by the Nrf2 inhibitor.
4. The amount of reduced gluathione (GSH) as well as the ratio to oxidized gluathione GSSG followed the same trend as glutathione reductase.
5. The changes in cell viability, as measured by the MTT assay, were less than 25% but significant. These followed the same trends.

What was not covered in the Zhang publication was reactive thiols of Nrf2 (in the featured image of this post) and the possibility of α-LA and/or GSH interacting with cadmium.

Mercury vs dihydro-lipoic acid in PC12 cells [5]

This study out of Hokkaido University, Sapporo, Japan and coauthors from the U.S. and Bangladesh used the reduced form of lipoic acid. PC12 cells can be cultured to have neuron like properties. Cells were treated with/without Hg2+ (0, 1.25, and 2.50 μM), DHLA (50 μM), and combinations of DHLA pre-treatment and Hg²⁺ for 48 hours.

1. In this study, DHLA did not improve viability. Hg decreased PC12 cell viability. DHLA almost completely mitigated the Hg loss of viability.
2. The same can be said for the reduced glutathione, GSH, content and the glutathione reductase activity.
3. Hg increased the number of cells undergoing apoptosis. DHLA reversed this action.
4. Protein levels of signalling pathways were examined in figure 4 with the addition of heme oxygenase protein levels.
5. Apoptosis markers were presented in Figure 5
6. Figure 6 examined total Hg concentrations in (A) the PC12 cells. Addition of DHLA decreased the average Hg content in the cells by about 3x. (B) The Hg in the culture media of the cells was not different.
   

These results are particularly exciting. Unfortunately we do not know if the Hg was bound to GSH, DHLA, or both..

α-lipoic acid against cadmium in rabbit brains [6]

Six-week-old male New Zealand white rabbits were given 30 continuous days of intragastric injections of

• Group I: control rabbit received demineralized water (Cd diluent).
• Group II: rabbits received cadmium chloride(Cd; 3 mg/kg body weight)
• Group III: rabbits received α-LA (100 mg/kg body weight)
• Group IV: rabbits administered Cd and α-LA as in groups II and III. α-LA treatment was given an hour after Cd administration. .

Twenty-four hours after the last α-LA injection, rabbits were slaughtered. The brains were excised rapidly from each animal, rinsed with an ice-cold physiological saline solution, and immediately frozen in liquid nitrogen.

In addition to Cd, brain levels of Cu, Zn, Fe, Ca, and P were also analyzed. The essential elements were at times significantly different but within 50% of one another. α-LA treatment reduced brain Cd by close to 50%. This is a really big deal. It suggests that α-LA, one way or another, removes Cd. The authors later speculated that small, stray, toxic amounts of other metals may be removed so that they do not redox cycle.

Here is the run down on oxidative stress parameters:

• MDA (nmol/g) hydro peroxides Cd increased 3x normal. α-LA brought down to just 2x control.
• TAC (g/dl) There were only small (<50%) changes in total anti-oxidant capacity. Jus α-LA increased TAC a small amount.
• GSH (mg/g) The changes were small, hovering around 14% with α-LA increasing and Cd decreasing.
• GST (U/g) Glutathione S transferase activity was decreased by about two thirds by Cd, and brought up to 2/3 control by α-LA.
• CAT (U/g) Only minor changes were seen in catalase activity. The mRNA transcripts were also not markedly changed by the treatments.
• GR (U/l) Changes in glutathione reductase activity were minor and less than 25% of the control.
• SOD (U/g) Changes in super oxide dismutase activity were also minor. Cd alone produced a sizable decrease in mRNA transcripts. There was a lot of variability within groups.
• GPx (U/g) Cd reduced glutathione peroxidase activity to half of the control. α-LA, with or without Cd, increased this activity to close to twice the control level. The GPx transcripts were also doubled by α-LA.
• Metallothionene 3 and Nrf2 transcripts were decreased by Cd but restored with α-LA.

While a lot of these changes are small but significant. Small changes add up to large benefits. Saleh and coauthors looked favorably upon α-LA as a detoxification agent.

“Oral supplementation of ALA has a beneficial effect in protecting against Cd induced oxidative neuronal damage in the brain of developing rabbits. It was primarily due to its antioxidant activity ensured via trapping active transition metals (Zn2+, Fe2+, Cu2+, and
Cd²⁺), restoring lipid hydro peroxides, sustaining antioxidant molecules (particularly GSH and GSH-dependent enzymes), and regulation of MT3 and Nrf2-dependent anti oxidant (CAT, SOD1, and GPx1) gene expressions.”

α-LA seems to be for real

Is α-LA a heavy metal chelator? We don’t know for sure. The PC12 and rabbit studies leave open this possibility. If part of the mechanism of α-LA involves increased synthesis of GSH, should one also supplement with GSH substrate amino acids? What about other dietary compounds that activate Nrf2?

References

1. Huerta-Olvera SG, Macías-Barragán J, Ramos-Márquez ME, Armendáriz-Borunda J, Díaz-Barriga F, Siller-López F. Alpha-lipoic acid regulates heme oxygenase gene expression and nuclear Nrf2 activation as a mechanism of protection against arsenic exposure in HepG2 cells. Environ Toxicol Pharmacol. 2010 Mar;29(2):144-9.
2. Macias-Barragan J, Huerta-Olvera SG, Hernandez-Cañaveral I, Pereira-Suarez AL, Montoya-Buelna M. Cadmium and α-lipoic acid activate similar de novo synthesis and recycling pathways for glutathione balance. Environ Toxicol Pharmacol. 2017 Jun;52:38-46. PubMed
3. Shi C, Zhou X, Zhang J, Wang J, Xie H, Wu Z. α-Lipoic acid protects against the cytotoxicity and oxidative stress induced by cadmium in HepG2 cells through regeneration of glutathione by glutathione reductase via Nrf2/ARE signaling pathway. Environ Toxicol Pharmacol. 2016 Jul;45:274-81. PubMed
4. Zhang J, Zhou X, Wu W, Wang J, Xie H, Wu Z. Regeneration of glutathione by α-lipoic acid via Nrf2/ARE signaling pathway alleviates cadmium-induced HepG2 cell toxicity. Environ Toxicol Pharmacol. 2017 Apr;51:30-37. PubMed
5. Binte Hossain KF, Rahman MM, Sikder MT, Hosokawa T, Saito T, Kurasaki M. Regulatory effects of dihydrolipoic acid against inorganic mercury-mediated cytotoxicity and intrinsic apoptosis in PC12 cells. Ecotoxicol Environ Saf. 2020 Apr 1;192:110238. PubMed
6. Saleh HM, El-Sayed YS, Naser SM, Eltahawy AS, Onoda A, Umezawa M. Efficacy of α-lipoic acid against cadmium toxicity on metal ion and oxidative imbalance, and expression of metallothionein and antioxidant genes in rabbit brain. Environ Sci Pollut Res Int. 2017 Nov;24(31):24593-24601. PubMed