X-Message-Number: 30804
Date: Thu, 5 Jun 2008 19:34:57 -0700 (PDT)
From:
Subject: BHA for cryopreservation
[Butylated hydroxyanisol (BHA) is highly effective in prolonging cellular
viability during cold storage. However poor brain uptake may be a limiting
factor for cryonics.]
Transplantation. 2007 Apr 15;83(7):948-53.
Inhibition of ERK1/2 activation by phenolic antioxidants protects kidney
tubular cells during cold storage.
Karhum ki P, Tiitinen SL, Turpeinen H, Parkkinen J. Finnish Red Cross
Blood Service, Helsinki, Finland.
BACKGROUND: Cold storage of tissues induces reactive oxygen species
(ROS), which contribute to cell injury. We have compared different
antioxidants in protection of renal tubular cells against hypothermia
injury and studied their effect on cold-induced mitogen-activated protein
(MAP) kinase activation. METHODS: Cultured renal tubular epithelial cells
(LLC-PK1) were stored in University of Wisconsin solution supplemented
with compounds tested for 16 hr at 4 degrees C. Release of lactate
dehydrogenase and cellular adenosine triphosphate were
measured. Activation of MAP kinases was determined by Western
blotting. Intracellular ROS were monitored with a fluorescent
probe. RESULTS: Cold storage resulted in a substantial loss of cell
viability. The simple phenol butylated hydroxyanisol (BHA) most
effectively prevented hypothermia-induced cell injury, whereas about
100-fold higher concentration of the polyphenol epigallocatechin gallate
(EGCG) was needed, although EGCG most effectively scavenged intracellular
ROS elicited by serum withdrawal. The MEK inhibitor U0126 and reduced
nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor
diphenyleneiodonium effectively protected the cells against hypothermia
injury. ERK1/2 was rapidly activated during chilling of the cells and
this was inhibited by BHA but not by EGCG. CONCLUSION: The results
suggest that chilling of renal epithelial cells induces ROS generation by
NADPH oxidase, which leads to rapid activation of the MEK-ERK1/2 cascade
and initiation of cell injury. This can be prevented by antioxidants.
PMID: 17460567
Anim Reprod Sci. 1997 Aug;48(2-4):269-78.
Motility of ram spermatozoa during storage in a chemically-defined diluent
containing antioxidants.
Upreti GC, Jensen K, Oliver JE, Duganzich DM, Munday R, Smith JF.
AgResearch, Diary and Beef Division, Ruakura Research Centre, Hamilton, New
Zealand.
The effects of five antioxidants--Vitamin E (VE), butylated
hydroxyanisole (BHA), n-propyl gallate (n-PG), deferoxamine mesylate
(Desferal) and catalase (EC 1 . 11 . 1 . 6)--on the maintenance of motility
of ram spermatozoa in a chemically-defined ram semen diluent (RSD-1) have
been evaluated. VE, n-PG and Desferal inhibited spermatozoal motility. The
relative inhibition (i.e., ratio of change in % motility over 24 h between
the treatment group and the corresponding control) at equimolar
concentrations (100 microM) of Desferal, VE and n-PG were 1.6, 1.8 and 3.6
respectively. BHA had no effect at 10 microM but at lower concentrations,
gave a slight improvement in motility in freshly diluted spermatozoal
samples and in those stored for 1 day at 15 degrees C. The addition of
catalase to RSD-1 was also ineffective in improving the motility of
spermatozoa. The lack of beneficial effects of the tested antioxidants
suggests that RSD-1 itself may destroy reactive oxygen species and the
antioxidant activity of RSD-1 components requires further study.
PMID: 9452879
Pancreas. 1996 Aug;13(2):166-72.
Improved insulin secretion of cryopreserved human islets by antioxidant
treatment.
Janjic D, Andereggen E, Deng S, Bartley C, Buhler L, Morel P,
Wollheim CB. Department of Medicine, University of Geneva, Switzerland.
The viability of islets of Langerhans prior to grafting is believed
to influence the clinical outcome of islet transplantation. To determine
whether oxidative stress occurs during the isolation-purification
procedure as well as during tissue culture and cryopreservation, we have
measured the glutathione redox state (GSH/GSSG) of islets. Human islets
were purified by standard techniques from organ donors, cultured, and
cryopreserved. Glucose-induced insulin release was monitored in parallel
during static incubations to assess the function of the islets. Cultured
human islets responded by a 2.2-fold increase in insulin release to a
glucose challenge. After cryopreservation the hormonal response was
lower. Immediately after islet isolation the GSH/GSSG ratio was 25.2 +/-
5.2, and it increased slightly to 32.0 +/- 6.1 after 1-3 days in tissue
culture. The GSH/GSSG decreased significantly after cryopreservation to
12.2 +/- 3.4, suggesting that the freezing and thawing procedures imposed
oxidative stress on the islets. To explore this hypothesis further,
cryopreserved islets were treated with the antioxidant butylated
hydroxyanisole (BHA). Islets exposed to BHA showed an improved
glucose-induced insulin release and had an increased insulin content. BHA
also protected the islets when they were exposed to alloxan, a free
radical generating agent. However, after cryopreservation, BHA treatment
did not modify the glutathione redox state. Although the BHA effect could
not be explained merely by a change in the glutathione redox state, it is
not precluded that redox changes of other cell components ameliorate the
glucose sensitivity of the beta cells. Further studies will be needed to
determine possible ways of improving islet cryopreservation with antioxidant
treatments and particularly, to validate the present observations by in
vivo experiments in the context of clinical islet transplantation.
PMID: 8829185
[BHA appears tto have limited effects on brain.]
Neurochem Res. 2000 Mar;25(3):389-93.
Reduction of brain antioxidant defense upon treatment with butylated
hydroxyanisole (BHA) and Sudan III in Syrian golden hamster.
Romero FJ, Rom J, Bosch-Morell F, Romero B, Segura-Aguilar J,
Llombart-Bosch A, Ernster L. Department of Physiology, School of Medicine &
Dentistry, University of Valencia, Spain.
Treatment with the antioxidant butylated hydroxyanisole (BHA) or the azo
dye Sudan III during two weeks led to changes in the brain enzymatic
antioxidant defense of Syrian golden hamsters. BHA was able to induce liver
superoxide dismutase (SOD) 2-fold but had no effect on the brain SOD
activity, whereas SOD activity was reduced to 50% in brain and remained
unchanged in liver with Sudan III. These two substances are known inducers
of DT-diaphorase and in fact this enzymatic activity was induced 4- and
6-fold in liver with BHA and Sudan III, respectively. However, BHA promoted
a significant 40% reduction, whereas no change was observed with Sudan III
in brain DT-diaphorase activity. Glutathione(GSH)-related enzymatic
activities were also assayed in brain and liver. No induction was observed
with BHA or Sudan III for any of the activities tested in hamster brain: GSH
S-transferase (GST), GSH peroxidase (GSH-Px) and glutathione disulfide
(GSSG) reductase (GR). Only 1.3- and 1.4-fold increases of GST and GR
activities were observed in liver and no change in any of these enzymatic
activities in brain with BHA; a partial limitation of permeability to BHA of
the blood-brain barrier may explain this results. Furthermore, Sudan III
promoted reductions in all these GSH-related enzymatic activities in brain
and liver. The possible explanations for these results are discussed.
PMID: 10761984
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