Inter-individual variation, seasonal variation and close correlation of OGG1 and ERCC1 mRNA levels in full blood from healthy volunteers

Ulla Vogel1,5, Peter Møller2, Lars Dragsted3, Steffen Loft2, Anette Pedersen4 and Brittmarie Sandström4

1 National Institute of Occupational Health, Lersoe Parkallé 105, DK-2100 Copenhagen, Denmark,
2 Institute of Public Health, University of Copenhagen, Panum Institite, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark,
3 Institute of Food Safety and Nutrition, Danish Veterinary and Food Administration, 19 Mørkhøj Bygade, DK-2860 Søborg, Denmark and
4 Research Department of Human Nutrition, Royal Veterinary and Agricultural University, 30 Rolighedsvej, DK-1958 Frederiksberg C, Denmark


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The mRNA levels of the nucleotide excision DNA repair gene ERCC1 and the base excision DNA repair gene OGG1 were quantified in 43 healthy volunteers in a dietary intervention trial as markers for the DNA repair capacity. Nine samples were collected from each subject over a period of 52 days. Sampling took place from January to May. The mRNA levels of OGG1 and ERCC1 correlated closely (r = 0.86, P << 0.0001) after normalization to either 18S ribosomal RNA or to ß-actin mRNA. The levels of OGG1 and ERCC1 mRNA were relatively constant within an individual with intra-individual correlation (R2 = 0.45–0.46) in a General Linear Model. The amounts of ERCC1 and OGG1 relative to 18S RNA were doubled in May compared with January. This coincided with an increase in the monthly influx of sunlight from 18 MJ/m2 in January to 242 MJ/m2 in May. The mRNA levels of both ERCC1 and OGG1 were positively correlated to the average daily influx of sunlight in the previous 30 and 5 days (r = 0.49; r = 0.37, respectively, P << 0.001). There were no significant effects of the dietary interventions. The inter-individual variation was 5–10-fold, which is more than the observed 2–3-fold seasonal variation. Thus, despite seasonal variation of the individual mRNA levels, the inter-person variation is still far larger than the intra-person variation, supporting the use as biomarkers.

Abbreviations: BER, base excision repair; NER, nucleotide excision repair


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
DNA is constantly damaged, e.g. by oxidation, dimerization and alkylation of bases. The capacity and efficiency of the repair enzymes is thus of major significance for the risk of cancer. Indeed, increased cancer risk is conveyed by several genetic defects (1–3) and polymorphisms in DNA repair genes (4–10) as well as decreased nucleotide excision repair (NER) capacity assessed by host factor assays (11–14). Environmental factors that induce DNA damage also increase expression of the DNA repair genes both at mRNA and protein levels (15–17). This may neutralize effects on levels in tissues and cells used, e.g. for assessment of biologically effective doses, although the risk may still be important due to error prone repair. Some protective effects of, for example dietary components could be related to increased DNA repair capacity without an increase in the rate of damage. Accordingly, monitoring expression of DNA repair enzymes may be of great value in the study of risks and benefits of environmental factors. Advances in technology, including real-time PCR allow sensitive and reproducible measurement of small amounts of mRNA with relatively high capacity.

DNA damage is repaired by different DNA repair pathways depending on the nature of the damage. Oxidations and alkylations of the nucleobases are typically repaired by base excision repair (BER), whereas bulky adducts are repaired by nucleotide excision repair (18,19). Both pathways are complex with many enzymes working in succession, and are probably intimately connected with overlapping substrate specificity (20,21).

ERCC1 encodes a subunit of the endonuclease, which makes the incision 5' of the DNA damage in NER (22). ERCC1 forms a heterodimer with XPF (23). Previous studies have shown that the mRNA levels of ERCC1 correlates with the DNA repair capacity in various tissues (24–27). Studies of ERCC1 expression in cell lines indicate that ERCC1 expression may be inducible (28,29).

OGG1 encodes the 8-oxo-guanine-DNA glycosylase, which removes 8-oxoguanine from DNA as part of the BER pathway (30). OGG1 expression has been shown to correlate with the repair capacity of 8-oxo-guanine (31). The mRNA level of OGG1 has been shown to increase vastly in rat lungs after installation of diesel exhaust particles (15), indicating that OGG1 expression is indeed inducible. A study of five healthy human subjects has shown a relatively high repeatability of OGG1 expression in white blood cells (32). We therefore decided to quantify the mRNA level of ERCC1 as a marker of the NER capacity and the mRNA level of OGG1 as a marker of BER capacity.

Solar radiation is an important source of DNA damage for healthy humans. We have shown previously that DNA strand breaks in circulating mononuclear blood cells correlated with the solar radiation over time (33,34). In a large retrospective study encompassing blood samples from 308 healthy subjects increased HPRT and Bcl-2 mutations were found in lymphocytes obtained in the summer compared with other seasons of the year (35). Solar radiation may induce both DNA photoproducts repaired by NER and oxidation of bases repaired by BER, and several NER genes have been shown to be UV-inducible (36–38). It is therefore conceivable that sunlight could influence the steady-state mRNA levels of the DNA repair gene, and contribute to a putative seasonal variation of the DNA repair gene mRNA levels.

In the present study, we used a human biomonitoring study to investigate the repeatability of OGG1 and ERCC1 mRNA expression in circulating blood cells of 43 human subjects over a period of 58 days. The study period was from January to May allowing investigation of the influence of solar radiation on gene expression. During half of the study period the subjects received a carefully controlled diet with 600 g of fruits and vegetables, no fruits and vegetables or an antioxidant and mineral supplement. Being without significant effect on the DNA repair gene expression levels the diet intervention will not be considered in the present paper. The transcription levels of ERCC1 and OGG1 correlated strongly within individuals and over time, although there were seasonal changes, which correlated with changes in solar radiation prior to sampling.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Forty-three healthy non-smoking volunteers, 22 males and 21 females, aged 21–56 years, were randomly distributed into three groups. During 28 days of the 58 days study period the subjects were given a basic diet devoid of all flavonoid-containing foods, including fruits, vegetables, juices, teas and chocolate products. One group was additionally given 600 g of vegetables and fruit (apple, pear, orange juice, carrot, broccoli, onion and tomato), in proportion to the common choice of these food items in Denmark. The second group was given supplement tablets containing the same levels of vitamins and minerals calculated to be present in the 600 g of fruits and vegetables (Pharma Vinci, Denmark) as well as an energy drink containing the same amounts of carbohydrates. The third (control) group was given the basic diet, placebo tablets and the energy drink. Further details of the diets and supplements will be published elsewhere. The volunteers were recruited in four periods spanning the months from January to May. The three groups were equally represented in the four periods, which consisted of 3 days run-in with habitual diet, 25 days of intervention and 4 weeks recovery on habitual diet. Two blood samples were collected initially during run-in, seven during intervention (days 2, 9, 16, 24 and 25) and two post-intervention after 1 and 4 weeks.

mRNA quantification
Total RNA was purified from 1.5 ml EDTA stabilized full blood <4 h after sampling. Qiagen blood RNA isolation kit was used as recommended by the manufacturer yielding ~5 µg total RNA. The cDNA synthesis was performed on 200 ng RNA in 10 µl using Gold RT–RCR kit (Applied Biosystems, Denmark).

Quantitative PCR was performed on ABI7700 sequence detection system (Applied Biosystems) in Universal Mastermix (Applied Biosystems) using 100 nM Tagman probe and 200 nM primers. PCR was performed in triplicate.

OGG1: primers were OGG1 714F, 5'-aaa ttc caa ggt gtg cga ctg-3'; OGG1 796R, 5'-gcg atg ttg ttg ttg gag ga-3'; probe, 5'-FAM-caa gac ccc atc gaa tgc ctt ttc tct tt-TAMRA-3'.

ERCC1: primers were ERCC1 512F, 5'-ggc gac gta att ccc gac t-3'. ERCC1 576R, tag cgg agg ctg agg aac a-3'; probe, 5'FAM-tgt gct ggg cca gag cac ctg t-TAMRA-3'. 18S and ß-actin primers and probes were obtained from Applied Biosystems.

ERCC1, OGG1 and 18S RNA levels were quantified in separate tubes. The average standard deviation on triplicates was 15%. The standard deviation on repeated measurements of the same sample (the control) in separate experiments was 20%, indicating that the day-to-day variation of the assay was 20%. The probes and primers have been validated, and the PCR was shown to be quantitative over a range of 130-fold dilution. Independent PCR reactions of the same samples yielded a correlation coefficient of 0.86 (Figure 1Go). Negative controls (where the RNA was not converted to cDNA) and positive controls were included in all sets. The standard deviation of the normalized mRNA levels in the positive control was 20%.



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Fig. 1. Correlation between two independent PCR on the same cDNA.

 
The daily influx of sunlight was obtained from the Danish Meteorological Institute.

Statistics
The relationship between expression data and solar radiation estimated as flux 5, 10 or 30 days prior to sampling were analysed by means of regression, controlling for sex, diet assignment, study day and study period as fixed categorical factors, day of year 2000 as continuous predictor and subject identity as random factor using General Linear Model MANCOVA/MANOVA, (Statistica 5.5; Statsoft, Tulsa, OH).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
ERCC1 and OGG1 mRNA levels in full blood were quantified by real-time RT–PCR. The mRNA levels were normalized to 18S, as 18S has been reported to give the least variation (32,39). As ribosomal RNA constitutes ~70% or more of the total RNA, the normalization was indirectly to the amount of total RNA. There was a strong correlation between the mRNA levels of ERCC1 and OGG1 when the mRNA levels were normalized to 18S (Figure 2AGo). Ten per cent of the samples were in a separate experiment also normalized to ß-actin, yielding the same close correlation between the ERCC1 and OGG1 mRNA levels (Figure 2BGo). The ERCC1 mRNA level constituted ~1% of ß-actin mRNA level, which was similar to previously published levels in lymphocytes (27).



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Fig. 2. Correlation between the relative mRNA levels of ERCC1 and OGG1. (A) After normalization to 18S. (B) After normalization to ß-actin mRNA.

 
Examination of the mRNA measurements revealed a strong dependence on sampling date (Figure 3Go). Sampling took place from January to May, and the mean normalized mRNA level increased during March, April and May. The effect was most pronounced for ERCC1 (Figure 3AGo). During the same time span, the daily influx of sunlight in Copenhagen increased from 18 MJ/m2 in January to over 40 MJ/m2 in February, 83 MJ/m2 in March, 146 MJ/m2 in April to 242 MJ/m2 in May. The solar influx was calculated as a 1, 5 10 and 30 day average flux. Analysis of the data using General Linear Model showed that the intra-subject variation was limited with respect to expression of ERCC1 and OGG1 mRNAs, i.e. the intra-individual correlation was strong with R2-values of 0.45–0.46. In the further GLM analysis the measures of solar flux prior to sampling were significant predictors of the level of ERCC1 and OGG1. For ERCC1 the flux 30 days prior to sampling was the only independent predictor with a regression estimate of 0.25 U increase in mRNA per unit of solar influx, 95% confidence interval 0.16–0.35 after adjustment for subject identity, study period, study day, sex and day in year 2000. The flux 5 days prior to sampling was the only independent predictor of the OGG1 level with a regression estimate of 0.047 U increase per unit solar influx, with a 95% confidence interval of 0.028–0.067 after similar adjustment. The associations and the related regression estimates between ERCC1 levels, OGG1 levels and solar flux were unchanged after controlling for study period, diet assignment, gender and day of the year, the latter as a continuous variable. In the whole dataset there were highly significant correlations between solar flux 30 and 5 days prior to sampling and the mRNA levels of ERCC1 (r = 0.49) and OGG1 (r = 0.37), respectively (Figure 4Go). However, from the GLM model and Figures 3 and 4GoGo it was apparent that the seasonal variation contributed limited to the total variation.



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Fig. 3. Average mRNA levels of ERCC1 and OGG1 as a function of the sampling date. Blood samples were collected from 43 healthy volunteers in four trial-series. The average mRNA levels and standard deviation was calculated for all available samples taken on that particular day. Sample size was nine, seven, 13 and 11 for period 1, 2, 3 and 4, respectively. Occasional missing samples decreased sample size. The smallest sample consisted of 6 determinations (one missing sample in period 2). The days are numbered from January first. Days 1–31 are January; days 32–60 are February; days 61–91 March; days 92–121 April; days 122–152 May. (A) Average ERCC1 mRNA levels, (B) average OGG1 mRNA levels. Stars denote statistically significantly (P < 0.05) higher mRNA levels compared with the average of the first two measurements of the mRNA level of the same group of persons (paired t-test, two-sided).

 


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Fig. 4. Average mRNA levels of ERCC1 and OGG1 as a function of the average daily influx of sunlight. Pearson's correlation coefficient and the corresponding P-values are shown. (A) Average ERCC1 levels as a function of the average daily influx the previous 30 days. (B) Average OGG1levels as a function of the average daily influx the previous 5 days. In the statistical model, separate data on all the individuals are used instead of the average.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The mRNA levels of ERCC1 and OGG1 were found to correlate closely with a correlation coefficient of 0.86–0.91. One could suspect that the correlation was caused by a normalization bias, when the amounts of ERCC1 and OGG1 mRNAs were normalized to 18S. However, the same close correlation was also found, when OGG1 and ERCC1 mRNA levels were normalized to ß-actin. Besides, control experiments have shown that the PCR reactions are linear in the range that was used. Furthermore, the same close association has been shown previously between the mRNA levels of XPD and ERCC1 (correlation coefficient 0.89), whereas ERCC1 and XPD mRNA levels did not correlate with mRNA levels of XPB, XPC, XPF, XPG or CSB, which all seemed to be inter-correlated (27). The fact that ERCC1 mRNA levels correlate closely with OGG1 mRNA levels could indicate that these DNA repair genes are subject to regulation by common factors. This has not been reported previously although it could be expected, as many DNA-damaging agents, for example solar radiation, give rise to both oxidative damage (UV-A light) and pyrimidine dimers (UV-B light) (40), which are repaired by BER and NER, respectively.

The observed variation in this dataset was much lower within subjects than between subjects. Furthermore, a substantial part of the observed intra-individual variation was seasonal and could be explained by the solar influx in the period preceding sampling, even when controlling for study period and day of the year. Nevertheless, it cannot be excluded that other uncontrolled for seasonal factors were the actual cause. As diet was carefully controlled during part of the study period and as such had no influence it is unlikely that seasonal changes in that respect were responsible. The reason for testing the effect of solar influx was that it has been shown previously that ERCC1 and other genes in NER are UV-inducible (36–38,41) in various model systems. Moreover, UV-irradiation of human fibroblasts has been shown to increase the DNA repair capacity of the cells (37). Increased exposure to solar radiation is a probable cause of the observed seasonal variation as solar influx was a stronger independent predictor than day in the period. Seasonal variation with respect to the level of strand breaks and UV-C induced unscheduled DNA synthesis in human lymphocytes is subject to seasonal variation, and that this variation was linked to the amount of solar radiation prior to sampling (33). Furthermore, increased amounts of strand-breaks were observed in human lymphocytes after a 1 week vacation on the Canary Islands compared with before the vacation (42). Thus, there is strong evidence that sunlight induces DNA damage in lymphocytes, and the increased damage could induce DNA repair. However, the data also suggested some differences in regulation with the highest influence of ERCC1 and OGG1 levels by solar flux 30 and 5 days prior to sampling, respectively. Indeed, up-regulation of OGG1 by oxidative stress induced by diesel particles in rat lungs occurred within 5–7 days after exposure (15).

Increased mRNA levels of DNA repair gene may not necessarily reflect increased DNA repair capacity. We have, however, shown previously that the mRNA level of ERCC1 correlates with the DNA repair capacity of UV-damage in PHA stimulated primary lymphocytes (27) and that low DNA repair capacity was associated with increased risk of basal cell carcinoma among psoriasis patients (11). Likewise, others have shown that low OGG1 mRNA levels are associated with low repair of 8-oxo-dG (43), and that high OGG1 mRNA levels are associated with high repair of 8-oxo-dG (16,31). Thus, assuming that the expression of DNA repair genes is also associated with DNA repair capacity in this experiment, the differences in DNA repair mRNA levels between individuals and within individuals over time could have biological significance.

The present study indicates that the intra-individual variation is limited compared with the inter-individual variation in mRNA levels. Previously, the variation within and between subjects was measured in a small study with only five healthy non-smokers and three samples per person (32). That study also indicated that the intra-individual variation was small compared with the inter-individual variation. As ERCC1 mRNA levels correlate with the DNA repair capacity in human lymphocytes (24,27) this suggests that mRNA levels could be valuable as biomarkers for the DNA repair capacity in future epidemiological studies, although seasonal variation may need to be accounted for.


    Notes
 
5 To who correspondence should be addressed Email: ubv{at}ami.dk Back


    Acknowledgments
 
E.Holst is thanked for valuable advice concerning the statistics. Anne-Karin Jensen is thanked for excellent assistance in the lab. This work was supported by Danish Medical Research Council Grant 9600259 and `Health effects of six-a-day', a grant from the Danish Ministry of Health, Research Centre for Environmental Health's Fund.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received May 28, 2002; revised June 7, 2002; accepted June 7, 2002.