The effects of chronic diseases on plutonium urinary excretion in former workers of the Mayak Production Association.

The radiochemical analysis of plutonium activity in urine is the main method for indirect estimation of doses of internal exposure from plutonium incorporation in professional workers. It was previously shown that late-in-life acute diseases, particularly those that affect the liver, can promote accelerated rates of release of plutonium from the liver with enhanced excretion rates. This initial study examines the relationships of some chronic diseases on plutonium excretion as well as the terminal relative distribution of plutonium between the liver and skeleton. Fourteen cases from former workers at the Mayak Production Association (Mayak PA) who provided from 4-9 urine plutonium bioassays for plutonium, had an autopsy conducted after death, and had sufficient clinical records to document their health status were used in this study. Enhanced plutonium excretion was associated with more serious chronic diseases, including cardiovascular diseases and other diseases that involved the liver. These chronic diseases were also associated with relatively less plutonium found in the liver relative to the skeleton determined by analyses conducted after autopsy. These data further document health conditions that affect plutonium biokinetics and organ deposition and retention patterns and suggest that health status should be considered when conducting plutonium bioassays as these may alter subsequent dosimetry and risk models.

Introduction

The Mayak Production Association (Mayak PA) constructed in 1948 was the first facility to produce plutonium primarily for nuclear weapons in the former Soviet Union. During the first decade of operation (1948–1958), a considerable number of workers were exposed to high levels of plutonium, primarily through inhalation of industrial aerosols. Exposures continued in later years but were greatly reduced with the introduction of more effective hygiene measures [1]. The Mayak Worker cohort contains more than 25,000 workers and studies have shown dose-related increases in cancers particularly in organs where plutonium is preferentially deposited, specifically lung, liver, and bone [2-6]. The Mayak Worker cohort has become a major source of information on the risks of longer-term occupational exposures to radiation [7].

Epidemiological studies that predict the risks associated with radiation doses from internally deposited plutonium are dependent on dose estimates to specific organs and tissues being obtained. In many cases, dosimetry estimates are made during life from bioassay measurements of plutonium excreted in urine. From the excretion rates, systemic and organ dosimetry estimates can be obtained from biokinetic and dosimetry models. Such models that have been applied to exposed Russian nuclear workers include the Mayak Worker Dosimetry System-2008 (MWDS-2008) [8], MWDS-2013 [9] and MWDS-2016 [10] models. Prior analyses of data obtained from workers at the Mayak Production Association (Mayak PA), Ozersk, Russia, have demonstrated considerable variability in plutonium excretion rates [11]. While there are a number of possible sources of variability in urine bioassay measurements, as discussed by Kathren and McInroy [12], improved calculation methods for bioassay results have been applied to the recent Mayak worker dosimetry models [13].

Late-in-life diseases, particularly those that affect the liver, are known to affect plutonium excretion rates as well as the relative retention of plutonium between the liver and skeleton. In a group of Mayak workers with advanced, late-in-life liver disease, for example, plutonium excretion rates were greatly elevated over those without liver disease [14, 15]. These studies also showed a correlation between disease severity and a relative decrease in the fraction of systemic (non-pulmonary) plutonium found in the liver with a concomitant increase in the skeleton. It was suggested that as plutonium was being released from the liver, more plutonium entered the circulation and became available for excretion or uptake into skeletal tissues [16]. The diseases that were associated with the greater excretion rates and relative redistribution of plutonium between the liver and skeleton included alcohol-related liver diseases, other diseases associated with fatty degeneration of the liver, and primary and metastatic cancers of the liver [16].

The prior studies cited above considered mostly late-in-life diseases, most of which were acute in nature. The purpose of the present study was to determine if some chronic diseases, particularly those that might affect the liver, have effects on plutonium excretion rates and the relative distribution of plutonium between the liver and skeleton. For this study, a group of Mayak PA workers were selected who had chronic diseases confirmed from clinical records, had multiple plutonium urine bioassays over a range of years, and had an autopsy conducted where terminal plutonium organ contents were measured. These initial results suggest that chronic diseases that involve the liver are associated with longer-term elevated rates of plutonium excretion. Additionally, chronic diseases are associated with less retention of plutonium in the liver relative to that in the skeleton as determined after autopsy.

Materials and methods

Cases

The study was done using anonymized records and was approved by the Human Ethics Review Panel of the Southern Urals Biophysics Institute and in compliance with the laws of the Russian Federation. Fourteen de-identified cases were selected from the Mayak PA database of former workers that met the following criteria: 1) They had suitable work histories and clinical records; 2) they had submitted to 4–9 bioassays for urinary excretion of plutonium; 3) their general health and status of chronic diseases during the periods of their bioassays could be estimated from the clinical records; and, 4) a terminal autopsy was conducted with measurements made to estimate total body and organ plutonium contents. The autopsies provided additional information on the presence and severity of any chronic diseases. The exposure route for the workers was through inhalation of plutonium containing aerosols in the workplace.

The characteristics of the cases are described in Table 1 and include the primary work location(s), years of employment at the Mayak PA, year of death, the total body plutonium burden, and information on the urine bioassays for plutonium that include the number of bioassays, the age at first bioassay and the year range when the bioassays were conducted. The selected individual workers started their work at the Mayak PA from 1949–1964 and finished working from 1956–1992 with employment ranging from 4–28 years. The workers had worked in the plutonium processing plant and/or the radiochemical plant. These plants were described in detail by Vasilenko et al. [1]. Urine bioassay measurements were initially conducted from 1971–1987 and the last bioassay for each worker ranged from 1984–2008. Bioassay results taken within one year of death were not used in this study.

Table 1

Characteristics of selected cases.

ID	Work locationa	Work period	Year of death	Plutonium body content, kBq	Bioassay examinations
					Number of bioassays	Age at first bioassay	Years of bioassay examinations
2095	Pu	1949–1957	2009	2.9	7	46	1975–2008
3108	Rc	1953–1957	2014	1.2	7	46	1972–2006
3513	Pu	1957–1978	2009	6.2	5	52	1987–2002
6962	Pu	1957–1984	2010	4.7	8	47	1977–2002
7790	Pu	1957–1984	2009	4.6	4	43	1983–2008
8046	Rc	1954–1971	1989	7.0	6	61	1980–1989
8260	Pu	1949–1962	1993	6.0	6	63	1977–1990
8686	Pu	1949–1960	1989	7.5	6	54	1972–1988
6984	Pu	1949–1974	1986	2.7	9	47	1971–1986
9127	Pu	1959–1973	1992	0.6	5	62	1976–1989
9277	Pu+Rc	1948–1956	1988	32.0	7	47	1974–1985
9351	Rc	1964–1992	1992	5.8	7	36	1974–1989
9418	Pu	1953–1960	1993	14.2	6	56	1978–1993
9451	Pu	1956–1983	1986	19.4	8	43	1975–1984

aWork locations: Pu, plutonium plant; RC, radiochemical plant.

Determination of health status

Several sources of information were used to reconstruct the health status of the workers during the period that bioassays were conducted. The workers were observed as inpatients in the clinics when the urine bioassays were conducted. Medical examinations were also performed at this time, thus the clinical records corresponded with the individual bioassay measurements. The health information in the records included any clinical diagnoses, status and/or progression of existing disease and information on smoking and alcohol use. Autopsy records were also available and included cause(s) of death, status of prior disease(s), and histopathology findings. The plutonium contents of the entire body and selected organs and tissues were also available. Because it was previously reported that liver diseases can alter the systemic (extrapulmonary) distribution and excretion of plutonium in the Mayak workers [15, 16], particular attention was given to the occurrence and progression of liver diseases or other diseases, such as congestive heart failure [17], known to involve the liver.

The cases were assigned to a “health status” group based on their records and these categories were similar to those used in prior studies [15, 16]. Those cases assigned to the “Healthy” category usually died from acute events or from a late cancer that likely did not affect the liver during the periods that bioassays were conducted. Individuals in this group had a number of other chronic diseases that included: cerebrovascular diseases, acute cerebrovascular events, diabetes, some cardiovascular diseases, gastrointestinal tract diseases, bone tissue dystrophies (osteochondroses) and others. Individuals with significant primary or secondary liver diseases were not included in the “Healthy” status group. Those cases assigned to the “Ill” health status category included cases with chronic or malignant diseases and conditions that appeared to be aggravated by alcohol consumption. Overt liver and acute cardiovascular diseases were not included in this category. The “Severely Ill” health status category included chronic diseases such as cancer metastases to the liver, heart diseases (such as congestive heart failure) and alcohol-related liver diseases. The cases assigned to these three health status groups are presented in Table 2. One case (#9451) was initially assigned to the “Ill” category but was later reassigned to the “Severely Ill” category due to progressive hepatic cirrhosis. A summary of the characteristics of the health status groups is presented in Table 3.

Table 2

Diseases recorded in the clinical records during the period of bioassay examinations and assignments to “health status” groups.

ID	Bioassay period (yr)a	Diseasesb noted in the clinical records during bioassay examination period	Health Status Group
2095	1975–2008	Gastrointestinal disease, congestive heart failure, hepatic congestion.	Severely Ill
3108	1972–2006	None	Healthy
3513	1987–2002	None	Healthy
6962	1977–2002	None	Healthy
7790	1983–2008	Gastrointestinal disease, chronic lung disease	Ill
8046	1980–1989	Cardiovascular disease, congestive heart failure, alcohol-related liver disease	Severely Ill
8260	1977–1990	None	Healthy
8686	1972–1988	None	Healthy
8984	1971–1986	Hepatitis, cerebrovascular disease, cancer with late liver metastases	Severely Ill
9127	1976–1989	Stomach cancer, chronic lung disease with later lung cancer, congestive heart failure	Severely Ill
9277	1974–1985	Cerebrovascular disease, gastrointestinal disease	Ill
9351	1974–1989	Alcohol-related liver disease, congestive heart failure	Severely Ill
9418	1978–1993	Cardiovascular disease, pneumonia	Ill
9451	1975–1984	Liver cirrhosis, later stomach cancer	Ill, Severely Illc

aFrom Table 1.

bParticular emphasis on diseases that may have affected the liver.

cIn Ill group from 1975–1981, then in Severely Ill group from 1983–1984.

Table 3

Summary of characteristics of the health status groups.

Health Status Group	Total number of bioassays	Age range at initial bioassay	Initial bioassay period (y range)	Last bioassay period (y range)
“Healthy”	32	44–63	1972–1987	1987–2006
“Ill”	23	43–56	1974–1983	1976–1999
“Severely Ill”	36	36–62	1971–1983	1984–2008

Urine bioassays measurements for plutonium

Urine plutonium bioassays were conducted during intervals for each case as indicated in Table 2 and overall the bioassays were conducted from 1971 through 2008. For these assays, workers were typically housed as inpatients for 3 or more days with multiple 24-hour urine samples collected [13]. From 1971 to 1998, plutonium was measured using alpha-radiometic methods. From 1998 to 2008, the assays were done using more sensitive alpha-spectrometry methods. The radiochemical procedures and associated limitations and uncertainties for these methods have been described in greater detail [13, 18–20].

Autopsy measurements for plutonium

Samples were collected from each autopsy case that included lungs, pulmonary lymph nodes and extrapulmonary organs that included liver, bone, spleen, and kidneys for the purposes of radiochemical analyses. Wet ashing techniques were applied to the soft tissues and dry ashing to bones. Prior to 2000, the tissues were prepared and plutonium was measured by alpha-radiometry and after 2000 plutonium was measured by alpha-spectrometry [20, 21]. Usually only selected samples were taken from the skeleton and some soft tissues, including the liver, and total organ weights were not recorded in some of the autopsy records. Thus, the total organ masses were estimated as previously described [8, 21] with total organ masses relative to body weight obtained from “Reference Man” from the International Commission for Radiation Protection (ICRP) [22].

Determination of excretion coefficients

To correlate plutonium urinary excretion with health status, coefficients for urinary plutonium excretion were determined for each case as of the date of bioassay examination. The excretion coefficients were calculated as previously described [16] relative to the total body content of plutonium, K, and the systemic, non-pulmonary body content of plutonium, K, measured at autopsy. Briefly, K−coefficient of the nuclide excretion from the body as the ratio of mean plutonium activity in the daily volume of urine U to the postmortem total body burden, estimated from the autopsy data.

K—coefficient of the nuclide excretion from the systemic burden (excluding the lungs and lymph nodes) expressed as a fraction of plutonium systemic content excreted per day:

Where: Q and Q are plutonium body content and plutonium systemic content, respectively (in Bq). U is the mean content (in Bq) of plutonium in the daily samples collected during each examination period. To calculate the plutonium body burden or plutonium systemic content, Q and Q , the plutonium concentration in Bq/g was extrapolated to the organ masses from “Reference Man” [22].

Partitioning of plutonium between liver and skeleton determined after autopsy

The distribution of plutonium between skeleton and liver was determined as their relative percentage fractions of the systemic (non-pulmonary) plutonium content in organs.

Statistical methods

The mean excretion coefficient was calculated from the multiple bioassays conducted over time for each worker in each of the health status groups. The data for each of the groups are expressed as the mean ± SD and as geometric means and geometric standard deviations (GSD). Differences between mean values for each of the health status groups were tested for significance using the single-factor analysis of variance (F-test). When statistically significant, pairwise comparisons were done using the least significant difference criterion. Results were considered to be statistically significant when p < 0.05.

Results

Excretion coefficients

Some of the workers had bioassays conducted near the end of their employment period, but most bioassays were conducted after employment and thus after the period where plutonium inhalations would have likely occurred. As expected, the excretion coefficients were very low but did correlate with overall health status during the period when bioassays were conducted (Table 4). The lowest excretion coefficients, K and K, were found in the cases that were assigned to the “Healthy” group.

Table 4

Urinary plutonium excretion coefficients calculated for the entire body (K) and for the systemic, non-pulmonary compartment (K) for the three health status groups of former Mayak PA workers.

Health Status Group	Excretion coefficient	Arithmetic Mean ± SD (Ke×10−5 d-1)	Geometric Mean	Geometric SD	Range
					Min—Max
Healthy	Ke body	0.67 ± 0.34a	0.59	1.73	0.11–1.88
	Ke syst	1.11 ± 0.59 a	0.97	1.74	0.26–2.78
Ill	Ke body	1.05 ± 0.51 a	0.95	1.53	0.41–2.80
	Ke syst	1.36 ± 0.75 a	1.15	2.00	0.11–3.72
Severely Ill	Ke body	1.86 ± 0.58 a	1.78	1.35	0.97–3.68
	Ke syst	2.96 ± 1.12 a	2.76	1.46	1.38–5.74

aStatistically significant differences in mean values K and K for all three groups were noted at level p<0.05 (F-test 52.7).

In those with more severe chronic diseases the excretion coefficients, K and K, were progressively greater in the “Ill” group and in the “Severely Ill” group (Table 4). The “Severely Ill” group was assigned those cases with more severe primary and secondary liver diseases that included liver metastases, alcohol related liver diseases, and cardiovascular diseases that affected the liver, such as congestive heart failure that resulted in hepatic congestion (Table 2). Comparing the K and K in the “Healthy” vs. the “Severely Ill” groups, for example, the K and K were almost 3 times greater in the “Severely Ill” group compared with the “Healthy” group (Table 4). The highest single measurement of K was 3.68 ×10−5 d- 1 in an individual undergoing chemotherapy (ID 9127 in Table 2). The mean values of K and K were statistically significant between the groups using a single-factor analysis of variance (Table 4).

Partitioning of plutonium between the liver and skeleton

The relative increases in the K associated with increasing severity of chronic diseases (Table 4) were also associated with decreases in the relative distributions of plutonium between the liver and skeleton as determined at autopsy (Table 5). While the variations (standard deviations) of these measurements were rather large, the mean liver/skeleton ratios of the percent of systemic plutonium content decreased with increasing chronic disease severity.

Table 5

Correlation of systemic plutonium urinary excretion measured from in-life bioassays and relative plutonium distribution between liver and skeleton determined at autopsy for the three health status groups of former Mayak PA workers.

Health Status Group	Numberof cases	Years of death	Years of examination	Ke syst×10−5 d-1 (mean ± SD)	Relative distribution, % of systemic content, ± SD		Liver/Skeleton Ratio, ± SD
					Liver	Skeleton
Healthy	5	1989–2014	1972–2006	1.11 ± 0.59a (32)b	23.9 ±16.7	70.4 ± 16.0	0.74 ± 0.49
Ill	4	1986–2009	1970–1999	1.36 ± 0.75 a	18.6 ± 16.3	75.5 ± 9.8	0.24 ± 0.20
				(23) b
Severely Ill	6	1986–2009	1971–2008	2.96 ± 1.12 a	13.4 ± 9.5	80.4 ± 9.3	0.21 ± 0.16
				(36) b

aData from Table 4.

bTotal number of bioassays from Table 3.

Discussion

The data from this initial study have established that some chronic conditions that occurred during the life of the workers at the Mayak PA were associated with prolonged elevated excretion rates of plutonium. The plutonium excretion rates, relative to the total body contents of plutonium, were progressively greater with disease severity. Additionally, the relative contents of plutonium in the liver relative to the skeleton, measured at autopsy, were less with progressive severity of the chronic diseases.

Acute late-in-life diseases, particularly those that affect the liver, are known to be associated with increased excretion rates of plutonium [14-16]. This was attributed to more plutonium entering the circulation from the liver and being available for excretion in the urine with the remainder being available for deposition in other tissues, particularly the skeleton [16]. This is supported by the autopsy data in the present study where the relative liver/skeleton ratios of plutonium were less with progressive severity of the chronic and acute late-in-life disease, supporting observations from prior studies on the Mayak PA workers [14-16].

In the present study, chronic conditions were identified in the clinical records that were known to be associated with liver diseases. These conditions included alcohol-related liver diseases, cirrhosis, cancers metastatic to the liver, cancer treatments that may have affected the liver, and congestive heart failure. Congestive heart failure, for example, was reported in the clinical records in four (ID 2095; 8046; 9127; 9351, Table 2) of the workers in the “Severely Ill” category and is known to be associated with both the cause and the consequence of certain liver dysfunctions [17, 23].

The relative reductions in liver plutonium content were associated with more serious chronic diseases. Prior studies have associated these changes and differences with histopathologically-defined liver diseases in the Mayak PA workers that included fatty degeneration and hepatocyte dystrophy [16]. Relative reductions in liver contents of fallout plutonium in humans with liver diseases have also been reported [24, 25]. In experimental studies, reductions in liver tissue metals and trace elements have been reported in a model of non-alcoholic fatty liver disease and these reductions were often concomitant with decreases in total liver protein [26]. Various liver diseases and at various stages of disease may also alter biliary excretion of the plutonium and thus fecal excretion rates relative to urinary excretion.

In addition to overt liver disease, alcohol consumption is known to be associated with reductions in liver content of plutonium in humans [16, 25] as well as americium in beagle dogs [27] and baboons [28]. Additionally, ethanol is reported to increase americium excretion rates from the liver in baboons [28, 29] and thorium in an individual previously injected with the radio-imaging agent Thorotrast with a bioassay conducted the day after moderate alcohol consumption [29]. These observations suggest that in addition to chronic and acute liver disease, the consumption of alcohol in the absence of overt liver disease may also result in elevated excretion rates of radionuclides and should be considered when conducting a bioassay.

The workers in this study were exposed to plutonium compounds during their work at the plutonium and radiochemical plants at the Mayak PA. Plutonium incorporated into the body is known to cause cancers, primarily at the sites of deposition that receive the largest radiation doses that include the lungs after inhalation of plutonium aerosols, and liver and skeleton after systemic distribution. Large epidemiology studies of the Mayak PA workers have established radiation dose-related cancers in these organs [4, 5]. While possible plutonium-associated pathologies were not established in the present study, it is possible that some of the identified chronic diseases that were associated with increased rates of plutonium excretion may have been caused or aggravated by the radiation exposures. Regardless of the etiology of the chronic diseases noted in this study, plutonium excretion rates and the distributions of plutonium between the liver and the skeleton were affected by the severity of the chronic diseases.

There are some limitations to this initial study that should be noted. The clinical histories were recorded over a period of 37 years (1971–2008) and there were considerable changes in clinical practices during this period. The interpretation of the clinical histories and records and subsequent assignment to the health status categories was based in clinical judgment of the available records and thus somewhat subjective. This initial study was also limited by the number of cases that could be identified that met the criteria for entry into the study that included suitable clinical histories, multiple bioassays over time, and suitable autopsy records. Additionally, the bioassays were conducted over a period of almost 4 decades. During this time, there were substantial improvements in the methodologies and instrumentation for both plutonium measurements in urine from bioassays and tissues from autopsy that included transitioning from alpha-radiometry in the earlier years to more sensitive alpha-spectroscopy in the later years [10, 21, 30].

This study shows that plutonium excretion rates measured at bioassay depend to a great measure on the health status of the individual at the time of the bioassay. If there were ongoing diseases that may affect the liver, this could cause significant deviations in the results of the bioassay measurements. Biokinetic models are used to estimate radiation doses to organs based on results from bioassays and autopsy data. If these models, such as ICRP-67 [31], assume a healthy individual, the bioassay measurements for workers with chronic diseases may overestimate the body and organ doses. Because increased severity of chronic diseases were also found to be associated with reductions in liver relative to skeletal contents of plutonium, the use of liver plutonium contents obtained after autopsy might underestimate radiation doses using standard biokinetic models. Thus, not considering the health status of the workers when the assays are conducted could lead to either over estimates or under estimates of disease risk from radiation exposures.

In conclusion, this initial study of Mayak workers shows that the severity of some chronic diseases known to affect the liver are associated with increased plutonium excretion rates and relative decreases in liver plutonium contents. Thus, the health status of the individual should be considered at the time of in-life bioassay and chronic and acute diseases should also be considered when autopsy data are used. Further studies are warranted to better determine the relationship of excretion enhancements with specific diseases and to the shorter-term consumption of alcohol on bioassay results and subsequent dose calculations and cancer risk estimates.

10 Jul 2020

PONE-D-20-04522

The effects of chronic diseases on plutonium urinary excretion in former workers of the Mayak Production Association

PLOS ONE

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The reviewers raised several concerns, including missing experimental details (e.g., the plutonium analysis), that are minor but need to be addressed.  Reviewer 3 pointed out that “plutonium may have resulted in liver diseases, which then in turn reduces the storage of plutonium in the liver.” This possibility could at least be acknowledged in the Discussion section.

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Reviewer #1: This study aimed to explore whether some chronic diseases, particularly those affecting liver, could enhance excretion rates of plutonium, having considered fourteen cases from former workers at the Mayak Production Association. The authors found that more serious chronic diseases, i.e., cardiovascular and liver diseases, were associated to increased plutonium excretion as well as with less retention of plutonium in the liver relative to that in the skeleton as determined after autopsy.

Overall, the manuscript is well written, methods and results are presented clearly, and discussion, including statement of study limits, is sufficiently detailed.

Minor comments.

Line 88. Based on Table 1, the workers finished working from 1956-1992 (not 1999), thus employment ranges from 7-33 years.

Table 1. Please amend the typo on 11th line, last column: 1985, not 1085.

Reviewer #2: The manuscript is very well interested and offer important way in the filed of environmental health. I have some recommendation for authors:

1. INTRODUCTION

The introduction part of the manuscript is very well written.

2. MATERIALS AND METHODS:

Please added the type of epidemiological study design; Period and area of observation.

Line 74: How did you define the criteria for cases selected (based on literature review, clinical recommendation, etc..). Please explain and put in the manuscript.

Line 79: Please define the "the different health categories". Added the explanation.

I recommended put the Tables 1, 2 and 3 in the results part of the manuscript.

Line 149: Please added the short description of methodology for Urine bioassays and autopsy tissue measurements for plutonium.

3. RESULTS

Line 232: Rewriten the title of table 5: "Correlation of systemic plutonium urinary excretion measured from in-life bioassays and relative plutonium distribution between liver and skeleton determined at autopsy for the three health groups of former Mayak PA workers.

4. DISSCUSION

General comments: rewritten the discussion part in this way:

1. The most important findings of your results related to the aim of the study.

2. Comparison your results with similar studies in this filed of research.

3. Limitation of results, methodology as well the strengths of your results and used methodology approach.

4. Why is your study important for the environmental health science.

5. Conclusion

Need to be shorter.

Reviewer #3: This study investigates plutonium activity in urine for the indirect estimation of doses of internal exposure; however, acknowledging that liver diseases, that could result from plutonium exposure could also affect the excretion of plutonium. This study of 14 cases shows the effect of chronic disease on plutonium excretion and the relative distribution of plutonium between the liver and the skeleton. Workers provided 4-9 urine bioassays for plutonium, had clinical records of their disease history, and had an autopsy conducted after death. Although there is repeated measurement data, the data analysis is cross-sectional. There is a need of proper biostatistical analyses.

Major Revisions

1. The authors analyze the data as cross-sectional observations, not using repeated assessments of plutonium (4-9 bioassays) nor the repeated disease status measurements.

2. The observation that they found relatively less plutonium in the liver relative to the skeleton determined by analyses conducted after autopsy may be due to their cross-sectional analysis.

3. The authors state in their abstract that plutonium excretion increased with more serious chronic diseases, including cardiovascular diseases and particularly diseases that involved the liver. However, they did not show any association with cardiovascular diseases in the main body of the text.

4. Although they have longitudinal data, the authors did not investigate whether plutonium may have resulted in liver diseases, which then in turn reduces the storage of plutonium in the liver. In the introduction they state that chronic diseases are associated with less retention of plutonium in the liver relative to that in the skeleton as determined after autopsy. However, the ‘causal link’ could likely be: “plutonium in the liver results in more liver diseases. The authors need to take the time order of the measurements of plutonium and the intermediate and final disease statuses into account (see Table 2). Hence in this paper, risk and consequences of these risks (diseases) are turned around. The final disease becomes the risk factor. See also Discussion section, page 13, line 243-247.

5. Page 10, Iine 173. It is not clear why the plutonium burden is extrapolated to an ‘ideal’ organ mass taking the ratio of actual organ mass and the organ mass from a reference man.

Minor Revisions

1. Page 6, 114-116: ‘also available’ repeated twice. Improve the style.

2. Page 8, Table 2: ‘Health Group’ should read ‘Health Status’

3. Page 10: GSD is not defined.

4. Pate 11, line 195-198 belongs to the Method section, not the Result section.

Same on page 12, line 214-215.

5. Page 14, line 255: “americium” needs to be capitalized.

6. Page 15: ‘ICRP 67’ is not defined.

Reviewer #4: The present manuscript reports a well-designed and structured work related with the excretion of plutonium from the human body and the prevalence of chronic diseases in workers from plutonium and radiochemical plants. The manuscript presents unique excretion rate profiles and health conditions of the selected workers which is relevant to evaluate their occupational exposure over an entire work career.

Please considerer some suggestions and recommendations to improve the manuscript.

Is there any information available in the literature reporting occupational exposure to plutonium thorough the inhalation? If so, authors could briefly describe that information and the major findings of those studies.

In the experimental section please provide a brief description of the methodologies used to determine plutonium in the urine and in organs. Please also add QA/QC data.

It would be interesting to some the major findings in a graphic comparison the three group of workers considered.

Lines 46-47: Authors should present the major possible sources of variability and highlight how the improved calculated methods reduce that variability.

Lines 58-59: If possible, authors could highlight the most described diseases, which would draw more attention to the relevance of this study.

Lines 177-180: this information could be integrated at the end of the previous sub-section.

Table 2,3, 5:

The column "Health Group" should be renamed as Group or similar to be in line with Table 4 which seems more correct since among the three groups considered only one refers to healthy workers. Authors should use a uniform way to mention the groups.

Table 4:

1) the geometric SD is predominantly higher than the respective geometric mean (except for the severely Ill), so this parameter may not be the most adequate to describe the profile of Plutonium excretion. Thus, it would be interesting if the range (minimum - maximum) of values observed in the participants was presented.

2) if the statistically significant differences were observed for all the three groups of workers, then the letter "a" should be in the column of the arithmetic mean (and not only in the line of health group).

Table 5

1) the letters "a" and "b" should be added to the Ill and Severely Ill lines of Ke syst.

2) The ratio liver/skeleton should be added to the table

Lines 281-289: Authors should highlight the potential of including workers' personal sampling to breathable plutonium in airborne particulate matter during regular working hours to a more complete health risk assessment.

Please see also some suggestions to improve the style and grammar used:

The expression "particularly those that affect the liver" are repetitively used in the text. I suggest the authors to adjust the manuscript t to become less repetitive.

line 27, 30-31: please rephrase to become clearer

lines 63-65: please rephrase this sentence

Line 89: The workers had worked (...); please rephrase.

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Reviewer #1: Yes: Francesca Gorini

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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25 Sep 2020

Response to Reviewers and additional information requested from the Editor.

We thank the 4 reviewers for their comments and support of our work. We have tried to incorporate the suggestions from the reviewers and have added additional clarifications, some additional references, and extended and clarified some of the discussion points.

Editor Requests

1. Style requirements. We have complied with this request.

2. Participant consent and anonymization: We have now added this complete statement at the beginning of the Methods section. “The study was done using anonymized records and was approved by the Human Ethics Review Panel of the Southern Urals Biophysics Institute and in compliance with the laws of the Russian Federation.”

We have also indicated in the next sentence that the records had been “de-identified”; “ Fourteen de-identified cases were selected from the Mayak PA database…..”

3. Statistical Analyses. Additional information is now provided in the Methods sections.

4. Availability of the data. Access to the data is available only upon request in compliance with the “Data Access Agreement” from the Joint Committee on Radiation Effects Research (JCCRER). The JCCRER is a committee established by a bilateral agreement between the U.S. and Russian Governments, signed by the U.S. Secretary of State and the Foreign Minister for the Russian Federation. Under this agreement, requests for data can be directed to Dr. Sergey Romanov, Director of the Southern Ural Biophysics Institute, Ozyorsk, Russia at Romanov@subi.ru.

Responses to Reviewers.

Reviewer #1: We appreciate the supportive comments from this reviewer.

Minor comments: The minor errors were corrected. Thank you!

Reviewer #2: We agree that this manuscript advances the field of environmental health, particularly for those either exposed or potentially exposed to some internally incorporated radionuclides and perhaps some other heavy metals.

1. Introduction: The introduction part of the manuscript is very well written.

We have made some changes in the Introduction as suggested by the other reviewers.

2. Materials and Methods: Please add the type of epidemiological study design; Period and area of observation.

We have reorganized parts of the Materials and Methods and have added additional information that includes that the workers were observed as in-patients in the clinics where both health assessments and bioassays were conducted, thus the biophysical examinations correspond in time with the medical examinations (in Table 1). Some additional information was also provided in the statistical methods section that hopefully adds some clarity to the design of this retrospective study using work history, medical, bioassay and autopsy data.

How did you define the criteria for cases selected (based on literature review, clinical recommendation, etc..). Please explain and put in the manuscript.

We have added a new paragraph at the first of the Introduction that provides some general background on plutonium metabolism and toxicity. The criteria for selection of cases were determined based on the earlier studies [references #15, 16] and known other causes of chronic liver disease (such as congestive heart failure) as well as the availably of the necessary information and data to conduct the study. The necessary criteria are listed in the first part of the Materials and Methods and additional information is provided in this section, including additional references in this section and the Discussion on congestive heart failure. We do want to emphasize that while the entire Mayak Worker Cohort is quite large (about 26,000 workers), there are relatively few workers who fit the specific and necessary criteria defined in our manuscript.

Line 79: Please define the "the different health categories".

We have moved this sentence (former line 79) to the later section ‘Determination of health status’ (now line 114) where the health status categories are now defined and referenced in greater detail.

I recommended put the Tables 1, 2 and 3 in the results part of the manuscript.

These tables (1-3) mainly contain descriptive characteristics of cases whereas the excretion and autopsy data are the primary results (Table 4-5). Our preference is to leave the Tables in proximity to where they are first cited for the convenience of the reader.

Please add the short description of methodology for Urine bioassays and autopsy tissue measurements for plutonium.

The assays are now briefly described and references cited where the techniques are described in detail, see lines 158-163 for urine bioassays and the new section on Autopsy measurements, lines 165-174.

3. Results: The title to Table 5 has been changed as suggested.

4. Discussion: The Discussion has been reorganized as suggested with some additions to address the importance in this field and further discussion points raised by the Editor and other reviewers.

5. Conclusion: We have shortened and focused the Conclusion as suggested.

Reviewer #3:

1. The authors analyze the data as cross-sectional observations, not using repeated assessments of plutonium (4-9 bioassays) nor the repeated disease status measurements.

Additional information has been added to the Materials and Methods, including more information in the Statistics section. The reviewer is correct in that the data were analyzed cross-sectionally. While there were multiple bioassays done over time in each individual, the mean of the bioassay measurements were used for each worker in each health category (see revised Statistical Methods section).

2. The observation that they found relatively less plutonium in the liver relative to the skeleton determined by analyses conducted after autopsy may be due to their cross-sectional analysis.

The skeleton and liver contents were real measurements of Pu content done after autopsy and not calculated from biokinetic models from the in-life urine bioassay data. We should note that the estimated organ nuclide contents are commonly done in living individuals using various biokinetics models, but much of the foundation for these biokinetics models comes from actual measurements done at necropsy in animal studies or autopsies in human studies. One of the main purposes of the present study is to provide information on worker diseases that may influence the interpretation of these in-life bioassay measurements.

3. The authors state in their abstract that plutonium excretion increased with more serious chronic diseases, including cardiovascular diseases and particularly diseases that involved the liver. However, they did not show any association with cardiovascular diseases in the main body of the text.

We thank the reviewer for asking this question as we agree that this is a subject that is important and needs further emphasis, particularly since 4 of the 6 workers in the “severely ill” categories had also been diagnosed with congestive heart failure. The relationship of heart disease with liver disease and visa versa is recognized and we have provided an additional recent reference (ref #24) in the Discussion on the importance of the disease relationships between the liver and heart. The relationship of cardiovascular-liver disease and the retention of plutonium and other nuclides and metals is a topic of interest that we are going to pursue in future studies.

4. Although they have longitudinal data, the authors did not investigate whether plutonium may have resulted in liver diseases, which then in turn reduces the storage of plutonium in the liver. In the introduction they state that chronic diseases are associated with less retention of plutonium in the liver relative to that in the skeleton as determined after autopsy. However, the ‘causal link’ could likely be: “plutonium in the liver results in more liver diseases. The authors need to take the time order of the measurements of plutonium and the intermediate and final disease statuses into account (see Table 2). Hence in this paper, risk and consequences of these risks (diseases) are turned around. The final disease becomes the risk factor.

The purpose of this study was not to establish the etiologies of the livers diseases, rather to correlate disease severity with plutonium excretion. This is important to adequately estimate plutonium body content, calculate dose, and ultimately estimate cancers risks in living radiation workers or exposed members of the public. There were, however, indications of the etiologies of some of these diseases in the clinical records, such as congestive heart failure, metastatic disease and alcohol-related disease. However, the reviewer does have a valid point worthy of further discussion and clarification. Thus, we have added a paragraph in the Discussion (lines 288-298) that discusses the large epidemiological studies done in the Mayak Worker Cohort (>26,000 workers) and the excesses in cancers per absorbed radiation dose in the primary target organs that include the liver. Some of these references are also now included in the Introduction. We also raise the possibility, as noted by the reviewer, that radiation exposures may have contributed to some of the liver diseases, such as metastatic disease, that were noted in the clinical records. We should note that there are no biological/molecular markers that are definitive for Pu-induced cancers, thus the associations of radiation exposures and various diseases is a statistical exercise as presented in the epidemiological studies now cited in the revised Introduction and Discussion (new references #2-7).

5. It is not clear why the plutonium burden is extrapolated to an ‘ideal’ organ mass taking the ratio of actual organ mass and the organ mass from a reference man.

The historic autopsy protocols that were conducted at the Southern Urals Biophysics Institute for the workers of the Mayak Production Association did not include entire organ weights (which would almost be impossible for the entire skeleton, for example), rather only portions of organs for histopathological evaluations. Thus, the International Commission on Radiation Protection (ICRP) “Standard Man” was used for the liver and the skeleton mass was calculated as per the reference cited in the manuscript (reference #22).

Minor Revisions: The identified minor revisions have all been addressed in the revised manuscript. We appreciate those suggestions.

Reviewer #4:

We appreciate the supportive comments of the importance of our work.

Is there any information available in the literature reporting occupational exposure to plutonium thorough the inhalation? If so, authors could briefly describe that information and the major findings of those studies.

We appreciate this suggestion to help the general reader with this important topic related to radiation-induced cancers. We have added a new paragraph at the beginning of the Introduction as well as a new paragraph in the Discussion that presents some of this background with new citations where the interested reader will be directed to some of the current literature on this.

In the experimental section please provide a brief description of the methodologies used to determine plutonium in the urine and in organs. Please also add QA/QC data.

We now identify in the revised text in the Methods where the limitations, sensitivities, uncertainties and QA/QC of different assay techniques are described. Specifically, lines 160-163 and 168-174 in the Methods and additions to the paragraph on limitations, lines 299-309, in the Discussion.

It would be interesting for some to have major findings in a graphic comparison the three group of workers considered.

We considered different ways to present some of these data graphically, but felt that the presentation of these data in Table form were more informative and concise.

Authors should present the major possible sources of variability and highlight how the improved calculated methods reduce that variability.

This topic is very important and has been detailed extensively in prior publications, the most relevant of which are cited in this manuscript. We have revised the Methods (both Urine Bioassay and the new Autopsy measurements sections) as well as parts of the Discussion to help direct the reader to this literature.

If possible, authors could highlight the most described diseases, which would draw more attention to the relevance of this study.

We appreciate this suggestion and have addressed two aspects of this. First we have now included more background on cancers associated with plutonium exposures in human in the first paragraph of the Introduction. Additionally, we have provided additional information on the conditions (such congestive heart failure) associated with the observed liver diseases in the Discussion and a new recent reference (revised paragraph from lines 265-270).

Lines 177-180: this information could be integrated at the end of the previous sub-section.

Even though this is a very short section, these data for the liver and skeleton are derived from autopsy data whereas the data in the prior section refer to the calculations of the urine excretion coefficients derived from in-life urine bioassays. We prefer to keep these separate to avoid misunderstandings of how these different data were derived.

Tables: The column "Health Group" should be renamed.

The groups have now been consistently labeled and referred to as “Health Status Groups” in the Tables and the body of the manuscript.

Table 4:

1) the geometric SD is predominantly higher than the respective geometric mean (except for the severely Ill), so this parameter may not be the most adequate to describe the profile of Plutonium excretion. Thus, it would be interesting if the range (minimum - maximum) of values observed in the participants was presented.

We have now included the range in Table 4.

2) if the statistically significant differences were observed for all the three groups of workers, then the letter "a" should be in the column of the arithmetic mean..

Done.

Table 5

1) the letters "a" and "b" should be added to the Ill and Severely Ill lines of Ke syst.

Done

2) The ratio liver/skeleton should be added to table 5.

Done

Lines 281-289: Authors should highlight the potential of including workers' personal sampling to breathable plutonium in airborne particulate matter during regular working hours to a more complete health risk assessment.

Unfortunately, we do not have access to all of this information. However, we should mention that we have now included additional background information and references on some of the large epidemiological studies on the Mayak Worker Cohort where some aspects of the nature of the industrial aerosols resident in different plant locations and changes over time are considered.

Please see also some suggestions to improve the style and grammar used:

We have tried to improve the style and grammar as suggested.

We thank the reviewers for their constructive and helpful comments and we feel the manuscript has been greatly improved by these suggestions.

Submitted filename: Responses to Reviewers and Editors Sept 24.docx

Click here for additional data file.

28 Oct 2020

The effects of chronic diseases on plutonium urinary excretion in former workers of the Mayak Production Association

PONE-D-20-04522R1

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Reviewer #2: All comments have been addressed

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

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Reviewer #2: Yes

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Reviewer #4: The manuscript was greatly improved after the revision. All suggestions and clarifications were addressed.

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5 Nov 2020

PONE-D-20-04522R1

The effects of chronic diseases on plutonium urinary excretion in former workers of the Mayak Production Association

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