1 Laboratory of Cell Genetics, Vrije Universiteit, Brussels, Belgium and 2 Unit of Industrial and Environmental Toxicology, Université catholique de Louvain, Brussels, Belgium
Dear Sir,
In their recent paper Hengstler and collaborators have measured DNA single strand breaks (DNA-SSB) in peripheral mononucleated cells from 78 individuals with alleged occupational exposure to heavy metals, namely cobalt, cadmium and lead and 22 controls (1). They concluded that co-exposure to these metals induced a more than multiplicative effect on DNA-SSB and suggested that DNA damage would already be detected from cobalt airborne levels of 4 µg/m3. If correct, this conclusion would be alarming because this level is well below the current exposure limits recommended by different agencies to protect the health of workers, e.g. the American Conference of Governmental Industrial Hygienists (ACGIH) recommends a threshold limit value of 20 µg/m3 (TWA) (2).
Combined exposure to heavy metals is an important research topic with considerable implications for the health of workers. Assessment of genotoxic effects of a single metal in biomonitoring studies is already difficult due to the multiple cellular targets that need to be addressed. Therefore, assessing interactions between different metals requires very well designed studies with adequate measurements of several complementary and well-validated biomarkers of exposure and effects. The work described by Hengstler et al. combined some exposure biomarkers, but only a single biomarker of genotoxic effects, namely DNA-SSB, which detects reversible lesions. Several technical issues, which we explain in the following paragraphs lead us to ask for taking their conclusions with care.
First, the population under study is a cause of concern because of its heterogeneity and of the virtual absence of metal exposure in these workers. They were recruited from various plants with very diverse industrial activities but they are treated in the statistical analysis as being homogeneously and exclusively exposed to metals, which is questionable. They were apparently selected because of suspected exposure to cadmium but, overall, their cadmiuria was not significantly elevated compared with the general population (median value of about 0.5 and maximum of 3 µg/g creatinine; Figure 1). The same is true for cobalt exposure with only six workers showing a cobalturia >3 µg/g creatinine. We also noted that cobalt was not detectable in the air of the working place of 33 individuals, but these were apparently not excluded from the analyses (e.g. in Table I).
The next issues relate to the measurement of exposure and effects. Most of the statistical analyses rely on airborne measurements but it is not described how they were sampled and what these measurements actually represent (respirable or inhalable fraction?). How exposure to other relevant genotoxic substances could be excluded with high probability should be clarified.
DNA damage was measured by alkaline elution on isolated mononucleated cells that had been cryopreserved before measurement. No information is given on the duration of cryopreservation. The authors claim that this technique is appropriate for performing alkaline elution on human mononuclear cells but the reference given to support this statement deals with cryopreserved hepatocytes used for in vitro enzyme induction studies, not alkaline elution. We really have difficulties in accepting that what was measured after cryopreservation is a real reflection of the DNA damage present at the time of sampling. Our experience with cryopreservation of mononucleated cells shows that the DNA is unstable under these conditions and that the extent of damage increases with the duration of cryopreservation (3). It is therefore probable that DNA-SSB measured by Hengstler et al. were influenced by the procedure of cryopreservation and the duration of storage. Whether this procedure is appropriate to reliably measure DNA repair capacity is even more questionable. Moreover, the authors do not mention the use in their alkaline elution procedure of an internal standard that allows integrating possible inter-experimental variation. The title and summary mention DNA repair measurements but methodology and data are not provided.
Cellular integrity was assessed by the trypan blue exclusion method but the proportion of viable cells was not reported. Whether this method is the most appropriate and sufficiently sensitive to assess lymphocyte integrity is questionable also. Keeping these technical concerns in mind, we, however, noted that the authors found negative correlations between DNA damage and trypan blue exclusion. It can, therefore, not be excluded that a significant fraction of measured DNA-SSB was the result of cell death, conceivably caused by the different manipulations of isolation, cryopreservation and thawing. We also noted that trypan blue exclusion was not correlated with metal exposure and was not included as an independent variable in the multivariate regression analysis from which the effect of metal exposure emerged.
We also have a number of concerns as to the interpretation of the results. First, we noted that no effect of smoking could be detected with the methodology applied in this study, which does suggest a lack of sensitivity and/or specificity; especially as a clear effect of smoking has been reported previously by the same group with the same method (4).
Secondly, we were surprised to see (page 70) that the correlations between DNA-SSB and cadmium in air (R = 0.371) was reported as relatively low whereas the correlation of the same parameter with cobalt in air (R = 0.401) was very good!
There also is a misconception in the interpretation of correlation analyses when authors state that one parameter (e.g. DNA-SSB) is a more sensitive end-point for biomonitoring of effect because the correlation coefficient is higher (e.g. than trypan blue exclusion).
Next, we regret that the authors did not discuss the inconsistencies in their findings, e.g. concerning the influence of lead exposure. How could they reconcile the fact that no correlation was found between DNA-SSB and lead in air or in blood with the results of their logistic regression analysis, which indicates an influence of lead in air alone and in interaction with cobalt in air?
It is also difficult to understand why DNA-SSB would correlate better with airborne measurements than with the internal dose. The authors argue that airborne measurements better reflect recent exposure; while this might be correct for lead and cadmium that are cumulative toxicants, it is not for cobalt, which is at the centre of their hypothesis.
The authors also claim that they have detected more than multiplicative effects of metal co-exposure but we did not find the statistical evidence to support this statement.
Finally, as acknowledged by the authors, this study was explorative (in search of an effect of cadmium exposure) and the results emerged from a multitude of statistical tests, not from an a priori-formulated hypothesis. Therefore, as for any epidemiological study, caution should be exercised when interpreting these results and they can certainly not be used to prove an effect.
To conclude, we feel that this publication did not provide convincing evidence to support such an alarming conclusion.
Notes
3 To whom correspondence should be addressed Email: lison{at}toxi.ucl.ac.be
References
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