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Journal of Occupational & Environmental Medicine:
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Pathological Excretion Patterns of Urinary Proteins in Miners Highly Exposed to Dinitrotoluene

Brüning, Thomas MD; Thier, Ricarda PhD; Mann, Helmut MD; Melzer, Heinrich PhD; Bröde, Peter Dipl-Math; Dallner, Gustav MD; Bolt, Hermann M. MD, PhD

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From the Institut für Arbeitsphysiologie an der Universität Dortmund (Dr Brüning, Dr Thier, Dr Bröde, Dr Bolt); the Medizinische Klinik II of the Rheinisch-Westfälische Technische Hochschule Aachen (Dr Mann, Dr Melzer); and the Clinical Center NOVUM, Huddinge, Karolinska Institute and University of Stockholm (Dr Dallner).

Address correspondence to: Thomas Brüning, MD, BGFA at the Ruhr-University, Buerkle-de-la-Camp-Platz 1, D-44789 Bochum, Germany; e-mail bruening@bgfa.de.

Copyright © by American College of Occupational and Environmental Medicine

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Abstract

A cohort of 161 underground miners who had been highly exposed to dinitrotoluene (DNT) in the copper-mining industry of the former German Democratic Republic was reinvestigated for signs of subclinical renal damage. The study included a screening of urinary proteins excreted by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and quantitations of the specific urinary proteins α1-microglobulin and glutathione-S-transferase α (GST α) as biomarkers for damage of the proximal tubule and glutathione-S-transferase π (GST π) for damage of the distal tubule. The exposures were categorized semiquantitatively (low, medium, high, and very high), according to the type and duration of professional contact with DNT. A straight dose-dependence of pathological protein excretion patterns with the semiquantitative ranking of DNT exposure was seen. Most of the previously reported cancer cases of the urinary tract, especially those in the higher exposed groups, were confined to pathological urinary protein excretion patterns. The damage from DNT was directed toward the tubular system. In many cases, the appearance of Tamm-Horsfall protein, a 105-kD protein marker, was noted. Data on the biomarkers α1-microglobulin, GST α, and GST π consistently demonstrated a dose-dependent increase in tubular damage, which confirmed the results of screening by SDS-PAGE and clearly indicated a nephrotoxic effect of DNT under the given conditions of exposure. Within the cluster of cancer patients observed among the DNT-exposed workers, only in exceptional cases were normal biomarker excretions found.

Environmental contamination of soil and groundwater with dinitrotoluene (DNT) at former military production and training areas is a matter of major concern. 1 On the basis of results from animal experiments, 2,4- and 2,6-dinitrotoluene (the main isomers present in technical DNT) were described as “possibly carcinogenic to humans (Group 2B)” by the International Agency for Research on Cancer. 2

We reported on a cluster of 14 cases of renal cell cancer (12 of the clear-cell type; 2 of the chromophilic type) and 6 cases of urothelial cancer (5 of the urinary bladder, 1 of the renal pelvis) within a group of 500 underground mining workers having high exposure levels to technical DNT in the copper-mining industry of the former German Democratic Republic. 3 This concurred with the suspicion of human carcinogenicity of DNT, with the urinary tract as the target system, and called for further clarification. 3

Hong et al 4 published a 2-year bioassay of 2,4-dinitrotoluene (100, 700, 5000 ppm) in the diet of Charles River CD-1 mice. Tumors of the renal tubular epithelium (diagnosed as cystic adenoma, cystic papillary adenoma, cystic papillary carcinoma, and solid carcinoma) were observed in male mice, with incidences after 2 years of 0 of 24 in controls, 6 of 22 at the low dose, 16 of 19 at the middle dose, and 10 of 29 at the high dose. Two of 8 high-dose male mice examined at the 1-year point of the experiment also had renal tumors. In addition to tumor development, there were clear signs of dose-dependent toxic nephropathy in the treated groups. 5 This experimental study points to the interrelation of renal toxicity and renal carcinogenicity of DNT.

Based on this interrelationship, we decided to reinvestigate the cohort of underground miners who had been highly exposed to DNT in the copper-mining industry of the former German Democratic Republic. 3 We used methods of previous studies of nephrotoxic effects in occupational cohorts highly exposed to trichloroethylene, 6–8 including a screening of urinary proteins excreted by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and quantitations of the specific urinary proteins α1-microglobulin and glutathione-S-transferase α (GST α) as biomarkers for damage of the proximal tubule, and glutathione-S-transferase π (GST π) for damage of the distal tubule.

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Subjects and Methods

The retrospective survey was a reinvestigation of the previously decribed 3 cohort of 183 workers of the underground copper-mining industry of the former German Democratic Republic, where explosives containing technical DNT had been extensively used. Details of the working conditions, which at present are considered unacceptable, were reported in the earlier study. Also as already reported, 3 the occupational histories, including exposures to any type of hazardous chemicals, had been obtained by personal interview using a specifically designed questionnaire. In addition, data regarding smoking, former kidney disease, and renal disease and of cancer within the patients’ families were recorded. The levels of occupational exposures to DNT were rated semiquantitatively, and four exposure categories were formed. 3 Specifically, the miners were asked for acute symptoms due to DNT, such as “nitro-headache,” tiredness, taste aberrations, and muscle weakness, in connection with occupational exposures. Specifically, the pharmacological symptoms of nitro-headache were frequently reported in connection with intensive skin contact to DNT.

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Exposure Assessment

The exposures were categorized according to the types and durations of professional contacts with DNT. A semiquantitative ranking into the categories of low, medium, high, and very high exposure was considered a practical compromise under the conditions given. Details of the procedures of retrospective exposure assessment and derivation of these exposure categories can be found in the previous report. 3

Of the 183 exposed miners studied earlier, 3 161 agreed to participate in the new study and provided a urine sample. In addition, all of the previously investigated exposed miners with renal cell cancer (n = 14), and all but one of those with urothelial cell cancer (n = 5), agreed to participate. As derived from the previous exposure assessments, 3 the distribution of DNT-exposed miners participating in the present reinvestigation is included in Table 1.

Table 1
Table 1
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Assessment of Urinary Biomarkers for Renal Damage

On chemical damage to the nephronic system, proteins are excreted that may serve as biomarkers of renal toxicity, particularly in the preclinical stage. In the present investigation, which focused on renal tubular toxicity, a two-step procedure consisted of a general screening of the molecular size of excreted proteins by SDS-PAGE, followed by a specific quantitative assessment of α1-microglobulin and of the GST isoenzymes α and π.

Urine samples were collected in glass containers, and aliquots were transferred to polypropylene tubes with polyethylene screw caps. SDS-PAGE analysis was performed on aliquots with no additives. For GST analysis, 10-mL aliquots were supplied with 0.5 mL of buffer (1M tris(hydroxymethyl)-aminomethane–hydrochloride, pH 7.6, 20% bovine serum albumin, 1% Tween 20, 0.05% sodium azide) to prevent protein adsorption onto the plastic material of the tube walls. 9 After collection, the samples were frozen at −20°C, stored, and thawed once just before analysis.

Levels of total protein, serum and urinary creatinine, and serum urea were measured in the urine according to standard clinical routine procedures. 10–12 In addition to the quantitative parameters of urinary excretion of GST isoenzymes and alpha-1-microglobulin, the electrophoretic SDS-PAGE pattern of excreted proteins as a screening tool was also included. This method allows for a semiquantitative examination of the protein excretion patterns. Tubular damage leads preferentially to the excretion of proteins with molecular weights lower than that of albumin, whereas excretion of larger proteins points to glomerular damage. For electrophoretic screening of the protein excretion patterns, the urine samples were subjected to SDS-PAGE using special precast gels (the Phast System, Pharmacia, Freiburg, FRG) with very high resolution. 13 After adjusting the urine protein concentration to 600 mg/L, 40 μL of urine was mixed with 10 μL of SDS-buffer (5% SDS) and incubated for 30 min at 37°C; 1 μL of this mixture was transferred to a Phast-gel, and electrophoresis was performed at 10 mA. The gels were silver-stained and analyzed by laser densitometry using an Ultrascan XL laser densitometer and a specific Gel Scan XL computer program. The patterns of excreted proteins discriminated among glomerular, tubular, or mixed renal damage. 13,14 Examples of typical SDS-PAGE profiles have been shown previously. 7 The evaluation was always done by the same clinical nephrologist (H.M.), who was blinded to names, exposure level, and disease status of the individuals. Particular attention was paid to excretion of the 105-kD Tamm-Horsfall protein, which is identical to the immunosuppressive glycoprotein uromodulin. 15

The α1-microglobulin was analyzed using a nephelometric method (Boehringer-Mannheim Biochemica (Mannheim, Germany). GST α and π excretions in urine were quantified by enzyme-linked immunosorbent assay procedures. Human GST α and π were purified to homogeneity from liver and placenta, respectively, by using affinity chromatography. Polyclonal antisera were produced by repeated immunization of rabbits, and monospecific antibodies were purified by immunoaffinity chromatography. 16,17 The detection limit for the enzyme-linked immunosorbent assay procedures was 0.1 ng/mL, and purified human enzymes were used as standards. All samples were analyzed within 1 month after collection; no decrease in enzyme contents was noted during this period.

For practical reasons, we did not adjust the urinary excretion data for creatinine. The assessments were based on volume, as recommended by the Deutsche Forschungsgemeinschaft 18 in conjunction with analyses of specific weight of the urine. Samples were excluded from further analysis and evaluation if the specific weight was below 1.010 g/mL or above 1.024 g/mL.

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Results

Results of the screenings of the urinary protein excretion patterns by SDS-PAGE of the DNT-exposed cohort, including the cases of cancer of the urinary tract, are compiled in Tables 1 and 2. In Table 1, only a general distinction was made between protein excretion patterns that were “normal” (only an albumin peak in SDS-PAGE) and pathological (tubular and/or glomerular damage, including excretion of tubular proteins of lower molecular weight and glomerular leakage with excretion of higher molecular weight proteins). A straight dose-dependence of pathological protein excretion patterns (tubular and/or glomerular damage) with the semiquantitative ranking of DNT exposure is obvious. The Cochran-Armitage Trend Test was highly significant for the miners without cancer (one-sided exact P < 0.001), and it reached borderline significance for the pooled data of the renal cell cancer and urothelial cancer cases (one-sided P = 0.058).

Table 2
Table 2
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The data may be compared with those of two previously assessed groups of persons not having been in occupational contact with nephrotoxicants. In 100 controls who had no history of overt renal disease and were not occupationally exposed to nephrotoxic chlorinated hydrocarbons (trichloroethylene in particular), 11 (11%) displayed protein excretion patterns indicative of tubular and/or glomerular damage. 7 In another group of 40 office workers not occupationally exposed to chemicals, 6 (15%) showed pathological protein excretion patterns based on SDS-PAGE analysis. 8 These figures underline the dose-dependence apparent in Table 1. Most of the previously reported cases of cancer of the urinary tract, especially in the more highly exposed groups, demonstrated pathological urinary protein excretion patterns.

Table 2 distinguishes between the qualitative patterns of protein excretions, based on SDS-PAGE, related to the subgroups of DNT exposure. It is obvious that the damage induced by DNT is directed toward the tubular system. Also noteworthy is the appearance in many cases of Tamm-Horsfall protein, a 105-kD protein marker.

Table 3 presents the quantitative urinary excretion data of the biomarkers α1-microglobulin, GST α, and GST π. The data consistently demonstrate a dose-dependent increase in the biomarkers α1-microglobulin and GST α, thus confirming the results of the SDS-PAGE screening, and they indicate a nephrotoxic effect of DNT toward the proximal tubule under the exposure conditions given. By contrast, no such effect is seen on GST π excretion, indicating no nephrotoxicity to the distal tubule.

Table 3
Table 3
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The results for the cluster of cancer patients observed among the DNT-exposed workers 3 are given in Table 4. The identification of the cases by subject number is identical to the original numbering in Table 2 of the previous publication 3 to allow easier comparison. Only in exceptional cases were normal biomarker excretions found in this group.

Table 4
Table 4
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Discussion

The hypothesis underlying the present investigation was that nephrotoxicity of (technical) DNT, under the past-exposure conditions to this explosive in the copper-mining industry of the former German Democratic Republic, 3 could be one factor contributing to the renal carcinogenicity of DNT. This hypothesis was derived from experimental animal data (see Introduction). The data presented here show a clear dose-related effect of occupational exposure to DNT on the excretion of protein biomarkers in the urine, indicative of proximal tubular damage.

In view of the extreme exposure conditions encountered, with typical vascular pharmacological effects (nitro-headache) frequently reported, the nephrotoxicity of DNT seems to be a high-dose phenomenon. This also explains why previous studies did not report on the human toxicity of DNT and is consistent with the results of animal experimentation. 4,5 A long-term (2-year) cancer bioassay showing nephrocarcinogenicity in male mice (see Introduction) involved the oral administration of 2,4-DNT to CD-1 mice at concentrations in the diet of 0 (controls), 100, 700, or 5000 ppm. Histopathological signs of nephrotoxicity were dose-related in this experiment and coincided with the general toxicity of the test compound, expressed in terms of retardation of weight gain (≈10% in the mid-exposed group at 12 months) and lifetime shortening (at the high-exposed group). 4,5 Certain unique features of the nephrotoxicity of DNT are evident from the present data:

The quantitative effects on the urinary biomarker proteins observed here can be compared with those reported in persons occupationally exposed to high doses of trichloroethylene. 7,8 When excretion of GST α is taken as a marker of proximal, and GST π as a marker of distal, tubular damage, trichloroethylene was a specific toxicant of the proximal, not the distal, tubule. 8 This is consistent with the present data (Table 3).

Tamm-Horsfall protein was detected in 60 of 177 urine samples (34%) from the DNT-exposed cohort. This physiological protein is associated with the plasma membrane of the luminal cells of the ascending limb of Henle’s loop and of the distal tubules. 19,20 The meaning of increased excretion of this protein, with respect to subclinical kidney damage, is not clear at present.

The most widely accepted biomarker of renal damage by occupational toxicants is α1-microglobulin. In previously studied non-exposed groups, a mean excretion of α1-microglobulin in the range of 4 to 7 mg/L was noted. The DNT-exposed groups (Table 3) showed a dose-dependent increase, with a mean of 13.7 mg/L in the very highly exposed group. In principle, this is comparable with the results observed 8 in workers exposed to high doses of trichloroethylene.

Consequently, the nephrotoxic and nephrocarcinogenic effects of DNT should be part of the medical surveillance of the exposed workforce, including the analysis of biomarkers for subclinical renal damage.

In essence, the present results are consistent with the concept of cancer initiation 21 by DNT isomers and the subsequent promotion of renal carcinogenesis by selective damage to the proximal tubule, which is highly analogous to concepts recently developed of the renal carcinogenicity of trichloroethylene. 22

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Acknowledgments

This collaborative study was supported by travel grants for T.B., R.T., and H.M.B. from the Deutscher Akademischer Austauschdienst and Bonn-Bad Godesberg, and for G.D. from the Svenska Institutet in Stockholm.

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References

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2. International Agency for Research on Cancer. Printing Processes and Printing Inks, Carbon Black and Some Nitro Compounds. Lyon: IARC; 1996:309–368. IARC monographs, vol. 65.

3. Brüning T, Chronz C, Thier R, Havelka J, Ko Y, Bolt HM. Occurrence of urinary tract tumors in miners highly exposed to dinitrotoluene. J Occup Environ Med. 1999; 41: 144–149.

4. Hong CB, Ellis HV, Lee CC, Sprinz H, Dacre JC, Glennon JP. Subchronic and chronic toxicity studies of 2,4-dinitrotoluene. Part III. CD-1 mice. J Am Coll Toxicol. 1985; 4: 257–269.

5. Ellis HV, Hagensen JH, Hodgson JR. Mammalian Toxicity of Munition Compounds. Phase III. Effects of Life-Time Exposure. Part I. 2,4-Dinitrotoluene. Kansas City, MO: Midwest Research Institute; 1978.

6. Brüning T, Golka K, Makropoulos V, Bolt HM. Preexistence of chronic tubular damage in cases of renal cell cancer after long and high exposure to trichloroethylene. Arch Toxicol. 1996; 70: 259–260.

7. Brüning T, Mann H, Melzer H, Sundberg AGM, Bolt HM. Pathological excretion patterns of urinary proteins in renal cell cancer patients exposed to trichloroethylene. Occup Med. 1999; 49: 299–305.

8. Brüning T, Sundberg AGM, Birner G, et al. Glutathione transferase alpha as a marker for tubular damage after trichloroethylene exposure. Arch Toxicol. 1999; 73: 246–254.

9. Sundberg A, Appelkvist EL, Dallner G, Nilsson R. Glutathione transferases in the urine: sensitive methods for detection of kidney damage induced by nephrotoxic agents in humans. Environ Health Perspect. 1994; 102 (suppl 3): 293–296.

10. Fawcett JK, Scott JE. A rapid and precise method for the determination of urea. J Clin Pathol. 1960; 13: 156–159.

11. Heinegård D, Tiderström G. Determination of serum creatinine by a direct colorimetric method. Clin Chim Acta. 1973; 43: 305–310.

12. Waller KV, Ward KM, Mahan JD, Wismatt DK. Current concepts in proteinuria. Clin Chemist. 1989; 35: 755–765.

13. Kierdorf H, Melzer H, Mann H, Siebert H. Differentiation of proteins in polyacrylamide gels by a modification of silver staining for the Phast System and a laser densitometer. Electrophoresis. 1993; 14: 820–822.

14. Bazzi C, Petrini C, Rizza V, Arrigo G, Beltrame A, D’Amico G. Characterisation of proteinuria in primary glomerulonephritides. SDS-PAGE patterns: clinical significance and prognostic value of low molecular weight (“tubular”) proteins. Am J Kidney Dis. 1997; 29: 27–35.

15. Neβelhut T, Rath W, Grospietsch G, Weber MH, Kuhn H. Urinary protein electrophoresis profile in normal and hypertensive pregnancies. Arch Gynecol Obstet. 1989; 246: 97–105.

16. Sundberg AGM, Appelkvist EL, Bäckman L, Dallner G. Quantitation of glutathione transferase-pi in the urine by radioimmunoassay. Nephron. 1994; 66: 162–169.

17. Sundberg AGM, Nilsson R, Appelkvist EL, Dallner G. ELISA procedures for the quantitation of glutathione transferases in the urine. Kidney Int. 1995; 48: 570–575.

18. Deutsche Forschungsgemeinschaft. Creatinine as a reference parameter for the concentration of substances in urine. In: Biological Exposure Values for Occupational Toxicants and Carcinogens. vol 3. Weinheim: VCH Publishers; 1998: 35–44.

19. Marier R, Fong E, Jansen M, Hodson CJ, Richards F. Antibody to Tamm-Horsfall protein in patients with urinary tract obstruction and vesicoureteral reflux. J Infect Dis. 1978; 138: 781–790.

20. Sikri KL, Foster CL, MacHugh N, Marshall RD. Localization of Tamm-Horsfall glycoprotein in the human kidney using immunofluorescence microscopical techniques. J Anat. 1981; 132: 597–605.

21. George SE, Kohan MJ, Warren SH. Hepatic DNA adducts and production of mutagenic urine in 2,6-dinitrotoluene-related B6C3F1 male mice. Cancer Lett. 1996; 102: 107–111.

22. Brüning T, Bolt HM. Renal toxicity and carcinogenicity of trichloroethylene: key results, mechanisms, and controversies. Crit Rev Toxicol 2000; 30: 253–285.

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Single Malt Canuck

With its fiddle music, rolling highlands, and Gaelic, Cape Breton can seem like a chunk of Scotland transported to the northeastern end of Nova Scotia. To that, Canada’s East Coast isle has added single-malt whiskey making. About a decade ago, the late Bruce Jardine, a Cape Breton businessman, decided to bring whiskey-making equipment and expertise home from Scotland. The Glenora Distillery set out to turn the spring-fed waters of Maclellans Brook into North America’s first single-malt whiskey—that exclusive category reserved only for whiskies made from malted barley from a single distillery. Because the operation is in Canada, the company … could not technically call the tipple “scotch”. Otherwise, “we do everything just like they do,” says master distiller Ken Roberts.

The end result? Connoisseurs call eight-year-old Glen Breton Rare Canadian Single Malt Whiskey … a “rich yet smooth subtle dram” and “instant classic.” At about $75 a bottle, it is “flying off the shelves”. … Requests have come from as far away as Japan and Europe.

—From DeMont J. Overture. Maclean’s. 2001;114(7):9.

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© 2001 Lippincott Williams & Wilkins, Inc.

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