Institute of Legal Medicine and Rudolf Boehm Institute of Pharmacology und Toxicology, Härtelstr. 16-18, D-04107 Leipzig, Germany
Dear Sir,
Thank you very much for giving us the opportunity to reply to the letter of Drs Lison and Kirsch-Volders concerning our article Occupational exposure to heavy metals: DNA damage induction and DNA repair inhibition prove co-exposures to cadmium, cobalt and lead as more dangerous than hitherto expected. Carcinogenesis, 2003, 24, 6373. We appreciate Drs Lison's and Kirsch-Volder's effort to discuss the relevance of exposure to heavy metals and possible implications for the health of exposed individuals. Concerning the present letter of Drs Lison and Kirsch-Volders we do not agree with their major arguments.
Drs Lison and Kirsch-Volders wrote: The work described by Hengstler et al. combined some exposure biomarkers, but only a single biomarker, ... namely DNA-SSB which detects reversible lesions.
Our reply: further biomarkers have been studied in the same population. For instance Jung et al., Institute of Occupational Medicine, University of Mainz, determined lymphocyte proliferation as well as chromate resistance in the same individuals described in our article (1). Interestingly, Jung et al. observed a clear association between heavy metal exposure and these biomarkers. In addition, the group of Dr Kai Janssen (Biochemical Institute for Environmental Carcinogens, Grosshansdorf, Germany) measured repair activity of 8-oxoguanine in mononuclear blood cells of the same individuals. Janssen also observed an association with heavy metal exposure and his biomarker. A manuscript in an international journal with Janssen's data is in preparation. Our data presented in Carcinogenesis represent only a part of a large and well-designed study planned, supervised and extensively documented by experts from the Ministry. Thus, independent laboratories using different biomarkers observed an association with heavy metal exposure in the same population described in our article, which supports our conclusions.
Drs Lison and Kirsch-Volders wrote: Moreover, the authors do not mention the use in their alkaline elution procedure of an internal standard that would have allowed to integrate possible inter-experimental variation.
Our reply: as is stated in the paper we did use a standard to control for variation between experiments, as described in our ref. 22. In the section Materials and Methods, DNA single strand break analysis we wrote in our publication in Carcinogenesis: Normalized elution rates were calculated as a measure for DNA single strand breaks (22). In our original ref. 22 (2) we describe that a standard was used to correct for inter-experimental variation (page 5623, last paragraph of our cited publication in Cancer Reserch). Normalized means divided by a standard.
Drs Lison and Kirsch-Volders wrote: Whether this method (the trypan blue exclusion methodJGH) is the most appropriate and sufficiently sensitive to assess lymphocyte integrity is questionable also. 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, cryopreservartion and thawing.
Our reply: we believe that most experts would accept the trypan blue exclusion assay as a possible screening test for cellular membrane integrity. However, we did not rely exclusively on trypan blue exclusion. We also performed two-dimensional gel electrophoresis to control for possible unspecific effects. A typical result is shown in figure 4 of our article. We wrote in the Discussion: ... two-dimensional gel electrophoresis shows that there are no broad alterations of protein expression in cobalt-exposed individuals. If DNA-single strand breaks would be caused by cell death due to manipulations it is unlikely that this would result in the above mentioned unaltered protein expression patterns. It is well known that cell death rapidly leads to protein degradation. However, this was not observed by two-dimensional gel electrophoresis.
Drs Lison and Kirsch-Volders wrote: DNA damage was measured by alkaline elution on isolated mononucleated cells that had been cryopreserved before measurement.... 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 but not alkaline elution. We really have difficulties is accepting that what was measured after cryopreservation is a real reflection of the DNA damage present at the time of sampling.
Our reply: indeed, we originally optimized a technique for cryopreservation of hepatocytes (described in our ref. 34; using hepatocytes we established for instance a computer controlled freezing protocol including a shock cooling step to compensate for the latent heat of fusion released during crystallization, optimized cryoprotectants and several further parameters important for maintenance of cellular integrity). As mentioned in our article the technique of cryopreservation originally established for cryopreservation of hepatocytes is also suitable for cryopreservation of mononuclear blood cells for alkaline filter elution analysis. We compared DNA single strand breaks of freshly isolated and cryopreserved mononuclear blood cells (with and without additional incubation with ethylene oxide and exposure to ionizing radiation) and observed very similar data. Meanwhile our technique of cryopreservation of cells for alkaline elution analysis has been successfully applied by other groups, for instance by PD Dr Gerhard Fritz (Division of Applied Toxicology, University of Mainz, Germany) and Prof. Dr Beyhan Ömer (Istanbul University, Department of Biochemistry, Turkey). Besides the above-mentioned technical aspects it is critical to keep the cells on ice after thawing and transfer them immediately into an ice-cold lysis buffer containing EDTA. Please consider also the above-mentioned controls by the trypan blue exclusion assay and two-dimensional gel electrophoresis.
Drs Lison and Kirsch-Volders wrote: We 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 since a clear effect of smoking has been reported previously by the same group with the same method (4).
Our reply: it is not correct that the study (ref. 4 in orignal manuscript) cited by Drs Lison and Kirsch-Volders (3) has been performed by my group. It is also factually wrong that Doerjer et al. report a clear effect of smoking habits... with the same method. In contrast, Doerjer et al. compared 17 male non-smoking controls and 15 male smoking painters and did not observe a difference (page 172, figure 1). In addition, 47 male smoking and 33 male non-smoking workers with suspected exposure to halogenated aromatics were compared, also without observation of a difference between both groups (page 173, figure 3). Both observations do not support a clear effect of smoking. Furthermore, Doerjer et al. compared seven male smoking automobile mechanics and seven male non-smoking automobile mechanics and observed a difference that due to the small case number should be interpreted with caution. In the cited paper Doerjer et al. did not compare smokers and non-smokers without additional occupational exposure to genotoxic substances. They come to the conclusion: To obtain more information about the groups which we started with, the groups will be completed, especially with respect to smoking habits (page 173, third paragraph).
It should be noted that several studies have been performed to examine the influence of cigarette smoking on DNA single strand breaks in mononuclear blood cells determined by alkaline elution. These studies have been cited in our article in Carcinogenesis as refs 37, 41 and 42 (46). Similar results have been obtained also by other groups, for instance by Prof. Dr Walter Popp (Hospital Hygiene, University Medical Center, Essen, Germany). There is a consensus that the influence of cigarette smoking on elution rates of mononuclear blood cells is relatively small. Therefore, relatively large populations are needed to clearly show this effect. When we compared more than 100 smokers and non-smokers the influence of cigarette smoking could clearly be shown (original ref. 37). However, in smaller populations the influence of cigarette smoking on blood cells will probably not become obvious by alkaline elution analysis (in contrast to blood cells much higher levels of DNA-SSB can be detected in alveolar macrophages of smokers that are exposed directly to cigarette smoke). There has been a lot of discussion about alkaline elution and cigarette smoking. It is clear that most biomonitoring techniques have limitations and one limitation of alkaline elution is its relatively low sensitivity to detect DNA-SSB caused by cigarette smoking in MNC (for this purpose for instance determination of sister chromatid exchanges offers a higher sensitivity). Nevertheless, numerous studies have shown that alkaline elution allows sensitive and reproducible identification of exposure to a large number of genotoxic agents. A negative result by alkaline elution does not exclude a possible genotoxic exposure. However, in our Carcinogenesis article we present a positive result. Thus, it is not logic to argue against this positive result (concerning heavy metals) by pointing out that the biomonitoring technique has some limitations for other genotoxic agents.
Drs Lison and Kirsch-Volders criticize selection and number of exposed individuals: ... 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 analysis (e.g. in Table I).
Our reply: this is an advantage of our study! We have a wide range of exposure levels including zero level. The population for biomonitoring has to be defined a priori. Later, we never exclude individuals from analysis! Please note that we studied 78 workers and an additional control group.
Drs Lison and Kirsch-Volders wrote: ... trypan blue exclusion was not correlated with metal exposure and was not included as an independent variable in the multivariable regression analysis from which the effect of metal exposure emerged.
Our reply: trypan blue exclusion was not an influential parameter in multivariable regression analysis.
Drs Lison and Kirsch-Volders wrote: 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 center of their hypothesis.
Our reply: the correlation analysis (Spearman's rank-correlation test) of cobalt exposure and DNA single strand breaks is shown in Table I of our manuscript: cobalt in air correlates with DNA single strand breaks (R = 0.401, P < 0.001) and also the internal dose cobalt in urine normalized to creatinine correlates with DNA single strand breaks (R = 0.381, P = 0.001). Therefore, it is difficult to understand why Drs Lison and Kirsch-Volders believe that DNA-SSB would correlate better with airborne measurements than with the internal dose. Because of the small difference between R = 0.401 and R = 0.381? This is obviously not justisfied. We did not report about such a difference. In contrast, on page 70 of our manuscript we write: Surprisingly, a very good correlation was observed between cobalt air concentrations and DNA-SSB (P < 0.001; R = 0.401). Similarly, cobalt in urine correlated with DNA-SSB (P = 0.001; R = 0.381). This situation is surprising, since cobalt exposure in our study was relatively low (range: 010 µg/m3 air) compared with the TRK-value (the concentration that should not be exceeded due to German regulations) of 100 µg/m3.
Drs Lison and Kirsch-Volders wrote: Finally, as acknowledged by the authors, this study was explorative and the results emerged from a multitude of statistical tests, not from an a priori-formulated hypothesis.... To conclude, we feel that this publication did not provide convincing evidence to support such an alarming conclusion.
Our reply: as we already mentioned from a statistical point of view this study was explorative. Nevertheless, the possible influential parameters, confounding factors and endpoints have been defined a priori. It is generally accepted that an individual study is not sufficient to prove a new hypothesis, since confirmation by independent investigators is needed. Recently our article (7) has been cited by McMurray and Tainer in Nature Genetics (8), together with several other alarming articles. Instead of repeating the conclusions from our own article I will finish with a citation from McMurray and Tainer (8): "As convincingly shown in this new study by Jin et al. (2003) (9), very low (micromolar) concentrations of Cd2+ ions, similar to those that can accumulate in organisms, markedly increase mutation rates in yeast. These new results indicate that cadmium primarily causes toxicity by inactivating an essential DNA repair activity. ...Together, these two analyses (Jin et al., 2003; Thilly et al., 2003) (9,10) suggest that environmental agents may select or prevent repair of mutations that arise from natural endogenous processes rather than inducing mutations directly.... Alteration of key DNA damage response pathways may prove even more important than direct DNA damage by mutagens. ... Cadmium toxicity may therefore represent a new mechanism by which genomes can be destabilized, and this observation expands the definition of the term mutagen".
Notes
Email: jan.hengstler{at}medizin.uni-leipzig.de
References
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