Cryopreserved versus freshly isolated lymphocytes in human biomonitoring: endogenous and induced DNA damage, antioxidant status and repair capability

Duthie, Susan J.; Pirie, Lynn P.; Jenkinson, A. Mc E.; Narayanan, Sabrina

doi:10.1093/mutage/17.3.211

Cited by 66 publications

(48 citation statements)

References 21 publications

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“…When cells are exposed to stressful environments, e.g., cooling, heating, radiation, or desiccation, cellular functions may be impaired. It has been observed that for some cells, preincubation in broth or liquid medium for some time after exposure to the stress may help repair the injuries (1,8,15,23); however, others doubt its effectiveness (7). In the present work, preincubation did not improve the recovery of cryopreserved BCG cells from cryoinjury.…”

Section: Discussioncontrasting

confidence: 61%

Cryopreservation of Mycobacterium tuberculosis Complex Cells

et al. 2012

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Successful long-term preservation of Mycobacterium tuberculosis cells is important for sample transport, research, biobanking, and the development of new drugs, vaccines, biomarkers, and diagnostics. In this report, Mycobacterium bovis bacillus CalmetteGuérin and M. tuberculosis H37Ra were used as models of M. tuberculosis complex strains to study cryopreservation of M. tuberculosis complex cells in diverse sample matrices at different cooling rates. Cells were cryopreserved in diverse sample matrices, namely, phosphate-buffered saline (PBS), Middlebrook 7H9 medium with or without added glycerol, and human sputum. The efficacy of cryopreservation was quantified by microbiological culture and microscopy with BacLight LIVE/DEAD staining. In all sample matrices examined, the microbiological culture results showed that the cooling rate was the most critical factor influencing cell viability. Slow cooling (a few degrees Celsius per minute) resulted in much higher M. tuberculosis complex recovery rates than rapid cooling (direct immersion in liquid nitrogen) (P < 0.05). Among the three defined cryopreservation media (PBS, 7H9, and 7H9 plus glycerol), there was no significant differential effect on viability (P ‫؍‬ 0.06 to 0.87). Preincubation of thawed M. tuberculosis complex cells in 7H9 broth for 20 h before culture on solid Middlebrook 7H10 plates did not help the recovery of the cells from cryoinjury (P ‫؍‬ 0.14 to 0.71). The BacLight LIVE/DEAD staining kit, based on Syto 9 and propidium iodide (PI), was also applied to assess cell envelope integrity after cryopreservation. Using the kit, similar percentages of "live" cells with intact envelopes were observed for samples cryopreserved under different conditions, which was inconsistent with the microbiological culture results. This implies that suboptimal cryopreservation might not cause severe damage to the cell wall and/or membrane but instead cause intracellular injury, which leads to the loss of cell viability.

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Section: Discussioncontrasting

confidence: 61%

Cryopreservation of Mycobacterium tuberculosis Complex Cells

et al. 2012

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“…This utilized cryopreserved lymphocytes for DNA damage determination. It has been reported that freezing of lymphocytes does not increase DNA strand breaks as compared to those of freshly isolated lymphocytes, and that fresh and frozen lymphocytes responded almost identically to hydrogen peroxide (Duthie et al, 2002). Visual scoring system used in this study reported to be accurately corresponding to the data that are measured by computer image analysis in human lymphocytes (Collins, 2004), in equine lymphocyte (Marlin et al, 2004) and in canine and feline leukocytes (Heaton et al, 2002).…”

Section: Discussionmentioning

confidence: 54%

Oxidative stress status of highly prolific sows during gestation and lactation

Berchieri-Ronchi

Kim

Yan

et al. 2011

Animal

167

122

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Elevated oxidative stress is reported to be associated with pregnancy complications in highly prolific sows. Oxidative DNA damage and the antioxidant status were determined in blood samples collected during the course of gestation and lactation in multiparous sows. Blood samples were drawn from sows (n 5 5) on days 30, 60, 90 and 110 of gestation (G30, G60, G90 and G110, respectively), on day 3, 10 and 18 of lactation (L3, L10 and L18, respectively) and on day 5 of postweaning (W5). Lymphocytes were isolated from the fresh blood and cryopreserved in each time point. Lymphocyte DNA damage was analyzed by alkaline single-cell gel electrophoresis (comet assay) to determine the single-and double-strand brakes and endogenous antioxidant concentrations using an HPLC system with UV detection. The comet assay showed elevated (P , 0.05) DNA damage (between 38% and 47%) throughout the gestational and lactational periods than during early gestation (G30; 21%). Plasma retinol concentration was reduced (P , 0.05) at the end of gestation (G110) compared with G30. Plasma a-tocopherol concentrations also showed a similar trend as to retinol. This study indicates that there is an increased systemic oxidative stress during late gestation and lactation, which are not fully recovered until the weaning compared with the G30, and that antioxidant nutrients in circulation substantially reduced in the mother pig at G110.

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“…Cryopreservation at −80°C or in liquid nitrogen has been widely used for human samples in general and PBMCs in particular (18,21). However, cryopreserved PBMCs were unable to remove H 2 O 2 -induced DNA lesions (22). In addition, both treated and untreated cells displayed increasing levels of DNA damage during repair incubation after exposure (22).…”

Section: Introductionmentioning

confidence: 99%

“…However, cryopreserved PBMCs were unable to remove H 2 O 2 -induced DNA lesions (22). In addition, both treated and untreated cells displayed increasing levels of DNA damage during repair incubation after exposure (22). Therefore, several studies used fresh lymphocytes for DNA repair studies using the alkaline comet assay (23,24).…”

Section: Introductionmentioning

confidence: 99%

A Modified Alkaline Comet Assay for Measuring DNA Repair Capacity in Human Populations

Trzeciak¹,

Barnes²,

Evans³

2008

Radiation Research

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Use of the alkaline comet assay to assess DNA repair capacity in human populations has been limited by several factors, including lack of methodology for use of unstimulated cryopreserved peripheral blood mononuclear cells (PBMCs), insufficient control of interexperimental variability, and limited analysis of DNA repair kinetics. We show that unstimulated cryopreserved PBMCs can be used in DNA repair studies performed using the comet assay. We have applied data standardization for the analysis of DNA repair capacity using negative and positive internal standards as controls for interexperimental variability. Our standardization procedure also uses negative controls, which provides a way to minimize the interference of interindividual variation in baseline DNA damage levels on DNA repair capacity measurements in populations. DNA repair capacity was assessed in a small human cohort using the parameters described in the literature including initial DNA damage, half-time of DNA repair, and residual DNA damage after 30 and 60 min. We have also introduced new DNA repair capacity parameter, initial rate of DNA repair. There was no difference in DNA repair capacity between fresh and cryopreserved PBMCs when measured by the Olive tail moment and tail DNA. The use of DNA repair capacity parameters in assessment of fast and slow single-strand break repair components is discussed.

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Cryopreserved versus freshly isolated lymphocytes in human biomonitoring: endogenous and induced DNA damage, antioxidant status and repair capability

Cited by 66 publications

References 21 publications

Cryopreservation of Mycobacterium tuberculosis Complex Cells

Cryopreservation of Mycobacterium tuberculosis Complex Cells

Oxidative stress status of highly prolific sows during gestation and lactation

A Modified Alkaline Comet Assay for Measuring DNA Repair Capacity in Human Populations

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