Cancer Letters
106 (1996)
235-242
Effects of four trihalomethanes on DNA strand breaks, renal hyaline droplet formation and serum testosterone in male F-344 rats’ C.L. Potter%*, L.W. Changb, A.B. DeAngeloc, F.B. Danielb “National
Risk Management
Laboratory, US Environmental Pmtectinn Agency, 26 W M.L. King Drive, Cincinnati, OH 45268, USA Monitoring System Laboratory, US Environmental Protection Agency, Cincinnati, OH. USA Research Laboratory, US Environmental Protection Agency, Reseaxh Triangle Park. NC, US)?
bEnvironmental ‘Health Effects
Received
Research
30 April
1996; revision
received
10 May
1996; accepted 20 May
1996
Abstract
All four possibletrihalomethanes (THMs) containingbromineand chlorine, as well as perchloroethylene(PCE), were evaluatedfor their ability to produceDNA strandbreaks,a2u-globulinrich renaldeposits,andtestosterone changesin male F-344 rats.Ratsreceiveddaily equimolardoses(0.75 or 1.5mmol/kg)of THMs or PCE (1000mg/kg) in 4% Emulphorvehicle by oral gavagefor 7 days.No significantDNA strandbreakswereproducedby anyTHM or PCEtreatment.PCE treatmentproducedincreasedhyalinedroplet formationin renaltubules.However,all THM treatmentsreducedor efiminatedthe appearance of renalhyalinedroplets.All four THM treatmentsalsoproduceda decrease in serumtestosterone concentrations on day 7, which mightaccountfor decreased hyalinedropletformation.No significantincreasein cell proliferation.measured by [3H]thymidineincorporationin vivo, appearedin this l-week study. Keywords:
Trihalomethanes; DNA strandbreaks;Hyalinedroplets;a2u-Globulin;Testosterone
1. btroduction Trihalomethanes (THMs) form as by-products during chlorine disinfection of drinking water [l-3]. Chloroform is the predominant THM formed during * Corresponding author. Mail e-mail:
[email protected]
Stop 420. Fax: +l 513 5697105;
t This documenthasbeenreviewedin accordance with US Environmental Protection Agency policy and approved for publication. Approval does not signify that the contents necessarily reflect the view or policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
0304-3835/96/$12.00 PI1 SO304-3835(96)0433
Q 1996 Elsevier l-5
Science Ireland
chlorine disinfection as a result of reactions between chlorine and organic compounds (e.g. humic acids) present in the water. However, the brominated methanes, brornodichloromethane, chlorodibromomethane and bromoform, may appear, depending on the concentration of bromine in the water. Certain halogenated hydrocarbon chemicals have been found to produce tumors in male (but not female) rat kidneys as well as increasedaccumulation of a2u-globulin-rich hyaline droplets. Current data suggest that tumor formation secondary to hyaline droplet formation reflects a responsespecific to the male rat kidney rather than a more general carcino-
Ltd. All rights reserved
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et al. /Cancer
genie potential of test chemicals [4]. As a response specific to the male rat, it may be viewed as an interfering factor in renal carcinogenesis studies used for human risk assessment. Bromodichloromethane produced kidney tumors in both male and female F-344 rats when administered by daily corn oil gavage [5]. Since most renal carcinogens in rats appear to produce tumors in males more than females, this compound might provide useful mechanistic information with respect to renal carcinogenicity in rats. Chlorodibromomethane, on the other hand, did not produce evidence of carcinogenicity in male or female F-344 rats in an NC1 corn oil gavage study [5]. However, chlorodibromomethane induced tumors in livers of female mice and kidneys of male mice. Therefore, it may be useful to compare short term effects of chlorodibromomethane to those of the rat carcinogens bromodichloromethane and chloroform. Of the THMs used in this study, chloroform has been the most studied with respect to toxicity and carcinogenicity. The brominated THMs, but not chloroform, produced positive results in bacterial mutagenesis assays [6]. Chloroform has produced renal carcinomas in male Osbourne-Mendel rats, but not in females, when administered via drinking water [7]. This short term study was designed to provide information on early effects in male F-344 rats. Since THM-induced carcinogenicity is sex-specific, effects of THMs on sex hormones was also of interest. Positive controls included: (1) perchloroethylene (PCE) for a2u-globulin-rich droplet formation in the male rat kidney; (2) dimethylnitrosamine (DMNA) for DNA strand breaks in the kidney; and (3) diethylnitrosamine (DENA) as a known rat kidney tumor initiator. 2. Materials
and methods
2.1. Chemicals PCE, chloroform, bromodichloromethane, chlorodibromomethane and bromoform were purchased from Aldrich Chemical Co., Inc. (Milwaukee, WI). DENA, DMNA and bovine serum albumin (BSA) were purchased from Sigma Chemical Co. (St. Louis, MO). [3H]Thymidine was purchased from Amersham Corporation (Arlington Heights, IL). Emulphor (a
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polyoxyethylated vegetable oil; GAF Corp., Linden, NJ) was mixed with distilled water to form a 4% v/v homogeneous solution. Compounds were administered with 4% Emulphor (5 ml/kg). Dosing solutions were prepared at the beginning of the study and analyzed daily by gas chromatography for concentrations of test chemicals. No decrement in THM or PCE concentrations in dosing solutions appeared during the 7-day study. Corn oil has been found to significantly impede absorption of chloroform, dichloromethane, dichloroethane, and trichloroethylene from the gut [9]. Compared to water as vehicle, corn oil vehicle has been shown to influence toxicity of chloroform [7,8,10] and bromodichloromethane [ 1 I]. Therefore, corn oil may not be appropriate for toxicity studies of environmental chemicals found in drinking water.
2.2. Animals Male F-344 rats weighing 100-150 g were purchased from Charles River Laboratories, Inc., Portage, MI. The animals were placed two per cage with woodshaving bedding, and maintained on Purina Laboratory Chow ad libitum at 22°C and 4060% humidity under a 12-h light/dark cycle. Animal care was performed according to the guidelines of the National Research Council and the American Association of Accreditation for Lab Animal Care. After 1 week in quarantine, rats were weighed and randomly distributed into treatment groups. Each THM and PCE treatment group contained four rats, and the DENA and DMNA groups each contained three rats. THMs were administered daily by oral gavage at equimolar doses (0.75 or 1.5 mmol/kg body weight) in 4% Emulphor (5 ml/kg) for 7 days. The daily PCE dose was 1000 mg/kg (6.0 mmol!kg) in 4% Emulphor. Control rats received vehicle alone. The highest doses were selected on the basis of previous work in bioassays. DENA and DMNA were administered in single doses of 100 mg/kg and 150 mglkg, respectively. Rats were weighed to the nearest tenth-gram at the start of the chemical dosing. At necropsy the body and kidney weights were taken and each animal was examined for gross lesions. Blood was collected from the portal vein and allowed to clot at 4’C. Serum was collected and stored at -80°C for later analysis of
C. L. Potter
et al. /Cancer
testosterone. Serum testosterone was measured using a kit from Diagnostic Products Corporation (Los Angeles, CA).
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IO6 (I 996) 235-242
strand breaks by an alkaline unwinding
237
procedure
[131. 2.5. Statistical analysis
2.3. Histology and autoradiography Osmotic pumps (Alzet Corp., Palo Alto, CA) for infusion of 200~1 of [3H]thymidine (62-64 Ci/mmol, 1 mCi/ml) were implanted into rats 3 days prior to termination. Rats killed on day 1 did not receive osmotic pumps. Four rats from each THM and PCE group were killed on days 1, 3 and 7. All rats in the DENA and DMNA groups were killed on day 1. The left kidney was removed, decapsulated, sectioned, and immersed in 10% phosphate-buffered formalin solution. Sections were stored at 4°C until they were embedded in paraffin and sectioned at 0.005 mm for autoradiography or staining with Mallory’s Heidenhain solution. Stained kidney sections were scored for hyaline droplets. Autoradiography was performed using a modification of the method of Gude [ 121. Kidney sections were dipped into liquid emulsion (Kodak NTB2, Rochester, NY) and exposed for 4 weeks prior to staining. Nuclei containing six grains or more were identified as cells undergoing DNA synthesis (S phase). Autoradiography data were quantified by counting labeled and unlabeled cells in four animals from each group. Data are presented as the number of labeled cells per 1000. 2.4. DNA strand break assay At the day 1 necropsy, the right kidney from each rat was quickly removed, rinsed, and placed in 12 ml of ice-cold modified Seligmann balanced salt solution (SBSS; 0.13 M NaCl, 2.68 mM KCl, 17.8 mM Na-acetate, 0.36 mM NaH2P04*H20, 0.73 mM KH2P04, 5.55 mM D-glucose, 0.14 mM NaHC03, 0.017 mM ascorbic acid at pH 7.2) freshly supplemented with 10 mM dithiothreitol. The kidney was passed through a cold stainless steel tissue press ( 1.O mm diameter sieve holes) from Harvard Apparatus (Millis, MA) to remove most connective tissue. The homogenate was mixed gently with the same 12 ml SBSS and then filtered through eight layers of cotton gauze. One ml of the homogenate was pelleted at 100 x g for 5 min at 4°C resuspended in 1 ml ice cold PBS/20 mM EDTA, and analyzed for DNA
Statistical analysis was performed on weight, DNA strand break, autoradiography, and hyaline droplet data using Levene’s test for multiple comparisons. Differences in serum concentrations of testosterone were determined by analysis of variance followed by Fisher’s least significant difference test for multiple comparison of means. Differences between treatments were considered significant at P < 0.05.
3. Results 3.1. Body and kidney weights Rats treated with the highest doses of bromodichloromethane and chlorodibromomethane weighed significantly less and had increased relative kidney weights relative to control rats after 7 days (Table 1). There were no other differences in finai body weights or kidney weights among treatment groups. There were no differences in absolute kidney weights at any time after any treatment in this study. 3.2. Hyaline drgulet formation PCE treatment resulted in increased hyaiine droplet formation in rat renal tubules (Table 2). All THMs tested reduced or eliminated the appearance of tubular hyaline droplets. Bromoform appeared to have the least effect on hyaline droplet formation of any THMs tested. However, hyaline droplets were not apparent in the high dose bromoform group by day 3, and were significantly reduced in the low dose group by day 7. 3.3. DNA strand breaks Treatment of rats with the 4% Emulphor vehicle resulted in a fraction of 0.83 kidney double-stranded DNA remaining after alkaline unwinding (Table 3). None of the THMs or PCE increased the number of DNA strand breaks relative to vehicle control. DENA (100 mg/kg) and DMNA (I 50 mgkg) reduced the
238
C.L. Potter et al. /Cuncer
Table
1
Body
and kidney
235-242
weights
Treatment”
Vehicle control (5 ml&) Chloroform (0.75 mmol/kg) Chloroform (1.5 mmollkg) Bromodichloromethane (0.75 mmoI/kg) Bromodichloromethane (1.5 mmolikg) Chlorodibromomethane (0.75 mmol/kg) Chlorodibromomethane (1.5 mmol/kg) Bromoform (0.75 mmol/kg) Bromoform (1.5 mmol/kg) Perchloroethylene ( 1000 mgkg) DENA DMNA
Letter.7 106 (1996)
Day
3 7 3
3 7 3 7 3
3 7 3 7 3 7 3 7 3 7
(100 mg/kg) (150 mg/kg)
aMale rats were dosed with test chemicals in 4% Emulphor moved, weighed and prepared for histology. *Significantly different from control (P < 0.05) by Levene’s
Body weight
Kidney
k) (*SEM)
(8) (GEM)
Relative kidney weight (‘70 of body weight)
1.220 f 0.033 1.268 + 0.030 1.414 f 0.052 1.202 * 0.720 1.186 + 0.056 1.315 f 0.059 1.283 kO.101 1.194*0.063 1.370 ” 0.043 1.228 c 0.036 1.244 + 0.037 I .360 + 0.034 1.243 + 0.067 1.180 + 0.040 1.383 kO.051 I.161 + 0.036 1.273 f 0.037 1.368 + 0.025 1.228 + 0.045 1.193 + 0.062 1.419 2 0.046 1.219 f 0.073 1.254 + 0.059 1.413 f 0.067 1.245 f 0.042 1.264 k 0.057 1.390 + 0.039 1.220 + 0.050 1.342 f 0.056 1.378 r 0.109 1.195 2 0.026 1.295 r 0.038
0.849 0.855 0.813 0.877 0.797 0.805 0.921 0.828 0.860 0.906 0.846 0.831 0.919” 0.883 0.896 0.866 0.864 0.840 0.930 0.889 0.953 0.885 0.820 0.826 0.887 0.889 0.871 0.886 0.907 0.866 0.840 1.045
143.7 148.6 174.0 136.8 148.6 163.4 139.5 144.0 159.3 136.0 147.1 163.7 135.4 133.7 154.3 134.2 147.3 162.9 132.3 134.0 149.1 137.5 152.6 170.9 140.5 140.6 159.7 137.7 148.1 158.3 142.7 123.6
+ 4.2 2 6.0 k 5.4 f 4.6 k 3.4 f 7.0 2 5.8 2 5.4 ” 2.4 T 5.3 + 1.2 f 4.8 + 4.8 2 5.3 * 5.3 * t 4.9 + 4.3 5 3.8 f 5.9 + 3.6 T 5.8 * + 5.5 k 5.2 t 5.3 2 5.2 2 5.7 + 5.5 + 4.9 iz 6.6 k 7.9 + 4.8 i 3.6
by gavage.
After
test for multiple
fraction of double-stranded DNA to 0.79 and 0.55, respectively, relative to vehicle treatment alone.
weight
1, 3, or 7 days rats were killed
ic + t f + f f + f f + f
0.005 0.016 0.020 0.030 0.023 0.013 0.070 0.022 0.017 0.029 0.013 0.012 0.039 f 0.012 SC0.009* * 0.013 f 0.008 f 0.007 +_ 0.026 5 0.024 + 0.020% t 0.021 + 0.017 + 0.023 k 0.024 + 0.023 k 0.015 + 0.022 + 0.018 f 0.029 k 0.045 f 0.002
and the left kidney
was re-
comparison.
There were no significant differences in [3H]thymidine labeling between vehicle control rats and any other treatment groups.
3.4. Autoradiography 3.5. Serum testosterone Fig. 1 shows rates of renal cell proliferation as measured by autoradiography after [3H]thymidine infusion. Large amounts of labeling were noted in the high dose chloroform group at day 3 and chlorodibromomethane group at day 7, but large variation about the mean precluded statistical significance.
All THMs, at the high dose, produced significant (P < 0.05) decreases in serum testosterone concentrations compared to the vehicle control value (Fig. 2). Large variation about the means prevented conclusions of statistical significance for the low-dose
CL.
Potter
et
al. /Cancer
Table 2 Kidney hyaline droplet scores Treatment”
Day
Hyaline droplet score (mean f SEM)
Vehicle control (5 mYkg)
I 3 7 1 3 7 1 3 7 1 3 7 1 3 7 1 3 7 1 3 7 1 3 7 1 3 7
1.00~0.00 1.00*0.15 1.25 + 0.25 0.75 A 0.25 0.00 f o.oo* 0.00 f o.oo* 0.25 It 0.25* 0.00 + o.oo* 0.00 ” o.oo* 0.50 rt 0.29* 0.25 + 0.25* 0.00 * o.oo* 0.50 + 0.29* 0.00 * o.oo* 0.00 It o.oo* 0.75 rf:0.25 0.25 f 0.25* 0.00 * o.oo* 0.50 f 0.29* 0.00 f o.oo* 0.00 + o.oo* 0.75 f 0.25 1 .oo f 0.4 1 0.25 + 0.25* 1.25 + 0.25 0.00 f o.oo* 0.00 f o.oo* 3.00 * o.oo* 3.00 + o.oo* 2.75 + 0.25*
Chloroform (0.75 mmollkg) Chloroform (1.5 mmol/kg) Bromodichloromethane (0.75 mmol/kg) Bromodichloromethane (1.5 mmollkg) Chlorodibromomethane (0.75 mmol/kg) Chlorodibromomethane (1.5 mmol/kg) Bromoform (0.75 mmol/kg) Bromoform (1.5 mmol/kg) Perchloroethylene (loo0 m&g)
1
3 7
aMale rats were. dosed with test chemicals in 4% Emulphor by gavage. After 1, 3, or 7 days, rats were killed and the left kidney was harvested for histology. *Significantly different from control (P < 0.05) by Jxvene’s test for multiple comparison.
chloroform, chlorodibromomethane and bromoform groups. In general, a dose-related decrease in serum testosterone values was evident in THM-treated rats. However, the low-dose bromodichloromethane group had serum testosterone v&es reduced below the high-dose values. The significance of this result remains unclear. F’CE treatment had no observable effect on serum testosterone values in F-344 rats. 4. Discussion Many chemicals known to induce tumor formation
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106 (1996)
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39
in male rat renal tubules appear to exert their actions via DNA damage or increased cell proliferation. However, in the present study neither of these events emerged as likely mechanisms of early tumorigenic activity by any of the THMs administered to male F344 rats. Chloroform has generally produced negative or inconclusive results in in vitro genotoxicity assays [ 141 and the Ames Salmonella assay [ 14-161. However, the three brominated THMs produced positive results in bacterial mutagenicity stu&es [IS]. Chloroform and other THMs produced sister chromatid exchanges (SCEs) in human lymphocytes in vitro. and in mouse bone marrow cells in vivo [IT], but negative results were reported for SCEs in human lymphocytes f 181 and Chinese hamster ovary cells [ 191. Fujie et al. [20] reported that all four THMs produced bone marrow chromosome aberrations in male and female Long-Evans rats 12 h after intraperitoneal injection, and in males 5 days after oral administration. Females were not treated orally in the study reported by Fujie et al. [20]. Lack of significant production of renal DNA strand breaks in vivo by any THM in the present study indicates these compounds were non-genotoxic to rat kidneys in vivo. Data obtained from in vivo strand break analysis could arguably carry more weight than data from genotoxicity tests performed in Table 3 DNA strand break induction by THMs Treatmen?’
Fb (mean r SEM)
Vehicle Control ChlQzofofm Bromodichloromethane Chlo&ibmmomethane Bromofotm PercIWWhylene Dietbylnitmsamine Dim&yi&os&mine
0.83 0.87 0.77 0.81 0.81 0.81 0.79 0.55
f 0.02 f 0.01 i- 0.08 t 0.04 rt 0.04 i 0.01 3z0.003* f 0.02*
aMale rats were dosed with test chemicals in 4% Emulphor (5 ml/kg) by gavage. All THM doses were 1.5 mmollkg. After 1 day, they were killed and the right kidney was immediately used for DNA strand break assay. bF represents the fraction of double stranded DNA remaining after 45 min unwinding. *Significantly different from control (P < 0.04) by Levene’s test for multiple comparison.
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C.L. Potter et (11. /Cuncer
Vehicle
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106 (1996)
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Control
Chloroform
0.75
Chloroform Bromodichloromethane
1.5 0.75
Bromodichloromethane
1.5
Chlorodibromomethane
n
Day
3
Day
7
0.75
Chlorodibromomethane
1.5
Bromoform
0.75
Bromoform
1.5
Perchloroethylene
0
10
5
Number
15
of Positive
20
25
30
Ceils / 1000
Fig. 1. Autoradiography of renal cells following treatment with [3H]thymidine and THMs (0.75 mmolIkg or 1.5 mmol/kg) or PCE (1000 mg/kg, 6.0 mmobkg). Control rats received 4% Emulphor vehicle (5 ml/kg). Osmotic pumps for infusion of [3H]thymidine were implanted into rats 3 days prior to termination. Bars represent the mean number of cells per 1000 (iSEM) that registered a positive reading for 3H (i.e. at least six granules/nucleus). None of the mean values were significantly different (P < 0.05) from control values, as determined by Levene’s test for multiple comparison of means.
Vehicle
Control
Chloroform Chloroform Bromodichloromethane Bromodichloromethane Chlorodibromomethane Chlorodibromomethane Bromoform Bromoform
0.75 1.5 0.75 1.5 0.75 1.5 0.75 1.5
Perchloroethylene 0.0
0.5
Serum
1.0
Testosterone
1.5
2.0
2.5
3.0
(ngiml)
Fig. 2. Concentrations of serum testosterone from rats 7 days after treatment with THMs (0.75 mmoykg or 1.5 mmobkg) or PCE (1000 mg/kg). Control rats received 4% Emulphor vehicle (5 ml/kg). Asterisks indicate mean values significantly different (P < 0.05) from control values, as determined by analysis of variance and Fisher’s least significant difference test for multiple comparison of means.
CL. Potter et al. /Cancer
isolated cells, especially cells not derived from the target tissue. This suggests that any renal tumors produced by these compounds in F-344 rats probably involve a mechanism(s) other than tumor initiation via direct DNA damage. Increased cell proliferation has been postulated to cause tumor formation by stimulating clonal expansion of spontaneously initiated intermediate cells [21,22]. In the present study, no statistically significant changes in tubular cell proliferation, measured as [3H]thymidine incorporation into DNA, occurred following treatment with any THMs for 1 week, although the highest doses of chloroform and chlorodibromomethane produced high levels of labeling in the kidneys of three of the four rats. Despite ambiguous proliferation measurements in this and other studies [22-241, negative in vivo genotoxicity results from this study are consistent with the hypothesis that renal tumorigenesis induced by chloroform, and possibly the brominated THMs, involves cytotoxic damage followed by cellular regeneration [3,21,22]. Many chemicals that produce kidney tumors in male rats also produce an increase in a2u-globulin rich hyaline droplet formation [25-271 and a syndrome known as male rat nephropathy. a2u-Globulin constitutes a major urinary protein of hepatic origin in the male rat. The protein is readily filtered through the renal glomerulus and taken up by renal tubule cells where it is degraded by lysosomal hydrolytic digestion [28]. a2u-Globulin accumulation occurs in male rat kidneys when the amount of protein taken up by tubule cells either overwhelms the digestive capability of cellular lysosomes [28], or the protein becomes resistant to lysosomal attack after binding of a chemical [27-311. Disabled hydrolysis and intralysosomal accumulation of a2u-globulin leads to renal epithelial necrosis followed by cellular regeneration with concomitant tumor promotion. Decreased hyaline droplet formation following administration of THMs suggests that this class of chemicals probably does not interact with the a2u-globulin protein to an extent required to block degradation at the lysosomes. Production of a2u-globulin is under multi hormonal control with strict androgen requirement [32-351. Little, if any, a2u-globulin appearsin the serum of female rats or immature males [36]. All THM treatments in this study decreased serum testosterone
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concentrations. This raisesthe possibility that THMs may have removed a critical stimulus for a2uglobulin production and secretion from the liver by decreasing the levels of circulating testasterone in rats. Decreaseda2u-globulin release from the liver might lower the amount of a2u-globulin arriving at the renal tubules to levels the kidney can consistently remove via lysosomes. Also, testosteronehas been shown to reduce proteolytic enzyme activity in the male rat kidney 1371. Consequently, reduction in circulating concentrations of testosteroneafter treatment with THMs might relieve the renal lysosomes of an inhibitory factor, thereby permitting more effective degradation of a2u-globulin by lysosomal proteases.Decreasedcirculating concentrations of testosteronein male F-344 rats treated with THMs also raises the question of whether THMs may produce androgenic deficiency in male rats. Assessmentof androgenic statusof male rats will require further study. References Ill PI
I31
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[24]
[25] [26]
[27]
[28]
[29]
1301
[31]
[32]
[33]
[34]
[35] [36]
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