A critical review of two scientific papers and what it means to us
Known by its chemical name, polychlorinated dibenzodioxin (PCDD), dioxins are known be chemical hazards that are present in all parts of the ecosystem - land, air and water (1, 2). Although not intentionally produced, PCDDs are a byproduct formed during manufacturing process of compounds such as pulp/ paper and metal smelters in presence of chlorine donor and are released into the environment as air pollutants (1, 2). Dioxins consist of two benzene rings joined by two oxygen atoms and have 4-8 chlorine atoms attached to the benzene rings and are extremely lipophilic compounds that bioaccumulate in the environment and organisms (3, 4). The varying position of chlorine atom accounts for 75 congeners that differ in their physical and toxicological properties (3, 4). PCDDs exert their toxicity by binding the Aryl Hydrocarbon Receptor (AhR), which translocates to the nucleus where it interacts with Ah receptor nuclear translocator (Arnt) protein and binds certain response elements, especially on CYP1A1 gene, leading to transcription of downstream mRNAs leading to proteins that can cause further damage (5, 6). Congeners from tetra-dibenzodioxin (TCDD) to Octa-dibenzodioxin (OCDD) are of particular interest to scientists (7). As a result, these two compounds and their associated toxicity in particular are being analyzed in this review.
Evaluating acute TCDD effects
In the paper by Valic et al, two patients with extraordinarily high levels of TCDD were studied to investigate genotoxic effects in vivo by using three different assays: Micronuclei assay (MN), Sister Chromatid Exchange Assay (SCE) and Single- cell gel electrophoresis assay-comet assay (8). The source of TCDD was not identifiable and therefore in order to have a proper reference range, they included people with normal reference TCDD levels along with negative controls (people with no TCDD levels) (8). TCDD levels in all the patients were measured using high resolution gas chromatography (144,000 pg/g and 26,000 pg/g blood lipid for proband patients) (8). MN assay was performed using patient lymphocytes whereas SCE assay was done using whole blood and chromosomes were treated and stained with Giemsa (8). When MN assay and SCE assay were perfumed during highest TCDD levels, interestingly there was no increase in micronucleus frequency; rather when TCDD decreased, a significant increase in micronucleus was observed (8). SCE remained within reference ranges throughout (8). Lastly, the Comet assay was used to measure of DNA strand breaks in lymphocytes (8). The five categories corresponded to the amount of DNA fragments in the tail, indicating the level of damage (8). Even though one patient showed no significant increase in damage, the second patient had a transient increase initially that leveled to moderate levels later on (8). The scientists hypothesized a delayed and transient effect due to an indirect or secondary genotoxic effect to explain the scientific phenomenon (8).
Is this of relevance?
This study has several issues regarding the way they performed the experiment and the way they presented their study. The introduction does not provide real substantive background information; rather it merely states how different organisms react to TCDD in a variety of ways. There is no “big picture” presented that helps a reader place this paper in context and never allows one to fully grasp the concepts. Rather than just state genotoxicity in various organisms when exposed to TCDD, they should have also included other mechanisms of toxicity related to TCDD such as developmental and neurological malformations (9, 10). Also, there is no background information provided on the various techniques and their relevance or advantages in measuring genotoxicity (11-14). Lastly, the overall aim of the study is stated in one sentence and could be further elaborated.
Regarding the methods used in this study, TCDD measurements in blood were measured only once. This could lead to increased analytical variation and it is imperative analytical variation to be lower than biological variation to be able to detect real differences. Hence, it would be prudent to use averages (multiple recordings at a single point) as it would have provided with better estimations. However the three methods used in this study were excellent ways to measure genotoxicity (11-14). Due to their different end point measurements, it is extremely useful in giving a complete picture in terms of damage: if there is damage in the nucleus during replication, incorrect crossing over or double/single strand breaks in the DNA (11-14). This provides a great assessment idea of the level of DNA damage the cell has incurred (11-14).
The paper did in a poor job in explaining and discussing what the results from the experiments mean. No in depth explanation was provided and more research should have been done as the reference list is too short for a scientific paper. When discussing the possible reasons for indirect toxicity, the authors state that one patient took several drugs. This could have been a confounding factor as it was not mentioned previous to the study and was not controlled. As a result, it could lead to more severe drug-drug interaction and downstream metabolites could have unknown and unmeasured effects. The authors did in a poor job in discussing the possible hypotheses of an imbalance in homeostasis through receptor altercation which led to genotoxicity as the authors provide no scientific backing or providing detailed explanation. Finally, the paper was poorly organized because of the inappropriate length of introduction and the omission of a conclusion. Conclusions are critical in allowing readers to fully grasp the discussed topic by summarizing the results found and their interpretation.
Conversely, are highly chlorinated PCDDs mixtures (with no TCDD) toxic?
In a study done by Smith et al, two Spanish families with increased body burdens of highly chlorinated PCDDs (hexa-CCD-Octa-CDD) were analyzed for a period of time in order to determine if exposure through cooking oil led to human health risk (15). The authors were particularly interested in these two families because finding true risk pertaining to some highly chlorinated PCDDs was difficult due to contamination with other compounds, they are found at very low concentrations and most of the studies have been done on TCDDs (15). They wanted to analyze the body burden in the parents and children and rate of elimination over a period of 6 years (15). They also wanted to determine if HpCDD and HxCDD caused a qualitatively and quantitatively different immunological response compared to TCDD and thus measured various aspects of the immune system (15).
Members from both families were exposed in unrelated incidents to PCDDs through cooking oil stored in containers that were previously used for pesticides (15). To analyze their body burden of highly chlorinated PCDDs and immunological components, blood samples were measured (15). Due to a lack of true reference group for PCDD levels, German population numbers were used; for immunological reference, age matched control groups from Italian TCDD studies were used (15). PCDDs were measured using high resolution gas chromatography- mass spectrometry; surface receptors on peripheral blood mononuclear cells were studied using flow cytometry (15). It was determined that adults had a higher body burden after six years compared to children who were able to eliminate the highly chlorinated PCDDs at a higher rate (15). Thus, it was an interesting finding because children might not always be the most vulnerable population (15). Also, children exposed in utero had high PCDD levels providing further support for PCDDs bioaccumulative characterstic (15). When immunological components were measured, interestingly, no significant alteration in expression of certain markers such as T-cells, B-cells and intergrin receptors (and many more) was seen (15). This led the authors to hypothesize that no conclusive information could be drawn from the data due to confounding variables such as inter/intra-variability and complex congener mixtures that hindered their ability to reach a definitive conclusion (15).
Are these valid findings?
The authors of this paper did an outstanding job by providing a complete and detailed introduction that provided the necessary background information. They clearly stated their goal and their rationale behind it and in doing so stated the difficulty in studying specific aspects of toxicology and evaluating human risk assessment. By presenting other studies pertaining to the issue of this difficulty in extrapolating information based on PCDD studies, they provided a holistic “picture” which allowed the reader to truly appreciate their findings.
However, even when clearly stated and acknowledged, using reference populations that were different from the study limits the power of the study as it adds another layer of complexity in the total variability in regards to chemical exposure, metabolism and excretion. Also using reference populations exposed to TCDD regarding immunological changes when studying HxPCDD-OCDD raised concerns since the mechanism of toxicity of the varying congeners is not fully understood. As a result, any fold change in expression of immunological markers when compared to reference may not be an accurate measure. When analyzing their results, the authors performed a detailed and powerful statistical analysis such as Analysis of Variance (ANOVA) where they compared the age controlled reference group's change in immunological response to subject's response. This allowed the authors to appraise if any real and statistically significant change was present.. Lastly, the authors' acknowledgement of the shortcomings of their conclusion was very good. They stated that any change in immunological response was not necessarily pathological and thus could be due to confounding factors such as age and regional differences. However this did have negative implications as one should be cautious in using this study to reach any conclusion. In their conclusion, they precisely summarized their findings, unlike the previous study, and provided their interpretation of data and also stated alternate hypothesis, backed by scientific research that could open future research opportunities.
The Final Verdict?
In this review, the findings from two studies were presented on two separate “kinds” of dioxins. The first study determined indirect or secondary genotoxicity due to TCDDs. The second study aimed at determining if subjects exposed to high levels of highly chlorinated PCDDs rather than just TCDD presented with different physiological changes, especially immunogenic responses. While the first study was able to reach a conclusion, the study had several shortcomings as stated earlier. The second study on the other hand, despite being unable to reach conclusive findings, provided a complete and holistic “overall picture” that made their study robust.
Over the years several studies have analyzed dioxin toxicity, ranging from using animal models (16) to epidemiological studies that analyze human exposure and toxicity (17, 18). However human response to dioxin varies considerably and species variability accounts for a huge unknown factor. Also exposure in the environment is nearly always comprised of a mixture of congeners and this complicates the issue further (19). Being able to deduce which congener led to a specific toxicity is extremely difficult if not impossible and thus leads to the most important issue of whether dioxin toxicity is relevant and should be studied further (2). This uncertainty hinders government officials and regulators to reach any definitive conclusion as to whether normal PCDD body burden is toxic. Using information based on animal studies, extreme exposure to various congeners of PCDD, regulators have taken several protective measures such as labeling TCDD a human carcinogen (20,21) and controlling emissions of PCDD in the environment (1). However, long term impacts of baseline PCDD remain elusive (22). As a result of this uncertainty future research in Bantox should be focused on innovative analytical techniques and models to determine human toxicity.