Chem. Res. Toxicol. 1991, 4, 528-534
528
Effect of Bromine and Chlorine Positioning in the Induction of Renal and Testicular Toxicity by Halogenated Propanes Marit LAg,*>tErik J. Serderlund,t James G. Omichinski,t?' Gunnar Brunborg,t J m n A. Holme,t Jon E. Dahl,ti§ Sidney D. Nelson,ll and Erik Dybingt Department of Environmental Medicine, National Institute of Public Health, Geitmyrsveien 75,N-0462 Oslo 4,Norway, and Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195 Received February 11, 1991 A series of halogenated propanes were studied for renal and testicular necrogenic effects in the rat and correlated to their ability to induce in vivo renal and testicular DNA damage and in vitro testicular DNA damage. 1,2-Dibromo-3-chloropropane(DBCP) and 1,2,3-tribromopropane were most potent in causing organ damage in both kidney and testes. Extensive necrosis was evident a t 85 pmol/kg in kidney and a t 170 pmol/kg in testis. The dibromomonochlorinated analogue 1,3-dibromo-2-chloropropane was less organ toxic than DBCP and 1,2,3-tribromopropane, but induced more organ damage than the dichloromonobrominated analogues 1bromo-2,3-dichloropropane and 1,3-dichloro-2-bromopropane.Dihalogenated propanes were even less necrogenic. These observed differences in toxic potency between the halogenated propanes could not be explained by relative differences in tissue concentrations. The ability of the halogenated propanes to induce DNA damage in vivo correlated well with their ability to induce organ damage. However, DNA damage occurred a t lower doses and at a shorter period of exposure than organ necrosis. This indicates that DNA damage might be an initial event in the development of organ necrosis by halogenated propanes in general. Further, testicular DNA damage induced by the halogenated propanes in vivo correlated well with the DNA damage observed in isolated testicular cells in vitro, showing that toxicity was due to in situ activation. The numbers, positions, and the types of halogen substituents appear to be important determinants in causing DNA damage and necrogenic effects. The toxic potential of the halogenated > propanes was in the following order: 1,2,3-tribromopropane 1 1,2-dibromo-3-chloropropane
1,3-dibromo-2-chloropropane > 1,3-dichloro-2-bromopropane l-bromo-2,3-dichloropropane
> 1,2,3-trichloropropane
1,2-dibromopropane 2 1,3-dibromopropane 2 1-bromo-3-chloropropane. The most toxic analogues contain three halogens with a t least two vicinal bromines.
Introduction Short-chain halogenated alkanes have been used industrially as chemical intermediates, extraction solvents, degreasing compounds, and copolymer cross-linking agents. Members of this chemical class have also been employed as pesticides. Several of the compounds have been reported to be mutagenic and carcinogenic, and to cause acute toxic effects in the kidney, testis, and/or liver. Metabolic activation is required for these chemicals to exert their biological effects. Such metabolic processes include halogen elimination, oxidation, epoxidation, and/or conjugation (1-9). Of the halogenated propanes, the toxic effects of 1,2dibromo-3-chloropropane (DBCP)' on kidney and testis are well documented (IO),and this compound has been extensively studied in our laboratories (11-18). It has been demonstrated that DNA damage is observed early after adminitration of DBCP, at doses lower than those causing organ necrosis (11, 13). Furthermore, studies with specifically methylated and deuterated DBCP analogues demonstrated that DNA damage and organ necrosis may be related (11,13). On the basis of these findings and also *To whom correspondence should be addressed. National Institute of Public Health. *Present address: Laboratory of Chemical Physics, NIDDK, National Institutes of Health, Bethesda, MD 20892. Present address: Scandinavian Institute of Dental Materials, P.O. Box 70, N-1344 Haslum, Norway. 11 University of Washington.
as supported by subsequent studies (16,18), it was proposed that DNA damage is an initiating event in DBCPinduced renal and testicular necrosis. DBCP may be activated to reactive metabolites both by a P-450 and by a glutathione S-transferase pathway (7,8, 12,14,19-21). However, P-450 metabolism appears to be of minor importance for DBCP-induced toxicity in rat kidney and testis (11-13). Recent studies in vitro (12,14) and in vivo (8) indicate that DBCP is activated to toxic intermediates by glutathione S-transferase mediated conjugation, presumably through the formation of electrophilic episulfonium ions. To further elucidate the mechanisms of kidney and testicular toxicity, a series of di- and trihalogenated propanes were synthesized and tested. The present study examines how variations in position, number, and type of halogen substituents affects renal and testicular necrosis, renal and testicular DNA damage, and the tissue distribution of the halogenated propanes.
Materials and Methods Chemicals. DBCP (Figure 1) and 1,2-dibromo-4-chlorobutane were synthesized by published methods, and the purity was >98% as determined by gas chromatographic (GC) analysis (22). Other chemicals were obtained from the following sources: 1,2,3-trichloropropane, 1,2-dibromopropane, 1,3-dibromopropane, 1~~~
Abbreviations: DBCP, 1,2-dibromo-3-chloropropane; GC, gas chromatographic;DMSO, dimethyl sulfoxide;NAAC, normalized area above curve.
0 1991 American Chemical Society 0893-228~/91/2704-0528$02.50/0
Organ Toxicity of Halogenated Propanes
Chem. Res. Toxicol., Vol. 4, No. 5, 1991 529
1,2,3-Tribromopropane
( 1,2,3- Tr iBP) 1,2-Dibrom0-3-chloropropane (1,2-DiB-3-CP, DBCP)
Bar
1,3-Dibrom0-2-chloropropane (1,3-DiB- 3-CP)
1,3-Dichloro- 2- bromopropane (1,3- DiC- 2- BP) 1-Bromo-2,3-dichloropropane (1 - B- 2,3- DiCP)
1,3-Dichloro-2-bromopropanewas prepared by bromination of the appropriate dichloropropanol using the same procedure previously described for synthesis of l-bromc-2,3-dichloropropane (24). The resulting yellow oil was vacuum distilled (55-57 "C at 10 mmHg) to yield 0.88 g of a colorless oil (5.8 mmol, 58% yield). The final product was 295% pure by GC analysis: 'H NMR (CDC13, 300 MH,) 6 4.35 (1H, m; C-2H) and 3.97 (4 H, m; C-1 and C-3 methylene); EI-MS, m / z 115/113/111 ( M + - Br), 77/75 (M'+ - Br - HC1). Animals. Male rata (MOL:WIST, weighing 200-250 g) were used. The animals were given RMI(E) standard pelleted feed from Special Diet Services, England, and water, ad libitum. Nephrotoxicity. (A) Histology. Groups of five male rata were given a single ip dose of DBCP (85 and 170 pmollkg), 1,2,3-tribromopropane (85 and 170 pmollkg), 1,3-dibromo-2chloropropane (170, 340, and 510 pmol/kg), 1,3-dichloro-2bromopropane, l-bromo-2,3-dichloropropane (500,750, and lo00 pmollkg), 1,3-dibromopropane (500, 750, and 1000 pmollkg), 1,2-dibromopropane (750, 1000, and 1500 pmollkg), 1,2,3-trichloropropane (1000, 2000, and 3000 pmol/kg), or 1-bromo-3chloroproprane (1000, 2000, and 3000 pmol/kg) in DMSO (0.5 mL/250 9). Control animals received DMSO only. The animals were anesthetized with a combination of fentanyl, fluanisone, and midazolam ip (0.02,1.0, and 0.5 mg/100 g, respectively) 48 h after administration of the propanes; the animals were then weighed and exsanguinated by collection of blood in heparinized syringes through aortic puncture. The kidneys were weighed, fixed in buffered formalin, and embedded in paraffin, and sections of the kidneys were prepared and stained with hematoxylin and eosin. The severity of tissue necrosis was determined blindly by microscopic examination as described for other nephrotoxicants (25). The extent of tubular necrosis in the combined areas of renal cortex and the outer zone of the medulla was graded from 0 to 4+; grade 1+necrosis was characterized by necrosis of individual cells, or groups of cells in adjoining tubules; grade 2+ was characterized by necrosis of all or most cells in adjoining tubules, but with viable tubules separating groups of necrotic tubules; grade 3+ was characterized by a zone of necrosis situated in the inner cortex and subcortical zone of the medulla; and grade 4+ was characterized by extensive necrosis in both the cortex and subcortical zone of the medulla. (B) Clinical Chemistry. Plasma urea and creatinine were determined with a Model SMAC autoanalyzer. Testicular Histology. Groups of five male rats were given a single ip dose of DBCP (85 and 170 pmol/kg), 1,2,3-tribromopropane (85 and 170 pmol/kg), 1,3-dibromo-2-chloropropane (170 and 340 pmol/kg), l,3-dichloro-2-bromopropane(500 and 1000 pmol/kg), l-bromo-2,3-dichloropropane(500 and lo00 pmol/kg), or 1,2-dibromopropane (500 and 1000 pmol/kg) in DMSO (0.5 mL/250 9). Control animals received DMSO. The animals were anesthetized, as described above, 10 days after administration of the propanes. Their testes were removed and weighed, then fixed in Bouin's solution (75% saturated solution of picric acid, 20% formaldehyde solution, and 5% acetic acid), and embedded in paraffin. Sections were cut and stained with hematoxylin-eosin. The extent and severity of seminiferous tubular necrosis and atrophy was determined blindly, essentially as described by Kluwe (19): grade 1+,1-10%; grade 2+, 11-25%; grade 3+, 26-50%; and grade 4+, more than 50% of seminiferous tubules were necrotic and/or atrophic. In Vivo Renal DNA Damage. Kidney DNA damage was determined by an automated alkaline elution system (26),which is based on a technique developed by Kohn and co-workers (27). Rats were killed 60 min after the ip administration of the test substance and their kidneys removed. The kidneys were placed in ice-cold modified Merchant's solution (0.14 M NaCl, 1.5 mM KH2P04,2.7 mM KC1,8.1 mM Na2HP0,, 10 mM Na2EDTA,pH 7.4) and minced into pieces with scissors. Kidney nuclei were prepared by gently squeezing the kidney fragments through a stainless steel screen (0.4 mm), followed by filtrations of the crude preparation (26). The nuclei were loaded onto polycarbonate fdten. DNA was eluted (0.03 mL/min) with a solution containing 20 mM Na2EDTA,pH 12.50. Two-hour fractions were collected, and DNA was determined fluorimetrically with the Hoechst 33258 dye. The elution rates are expressed as normalized area above termed "NAAC values". When the (semilogarithmic) curve (B),
"
c
"a
1,2,3-Trichloropropane (1,2,3-TriCP) 1,2-Dibromopropane (1,2-DiBP) 1,3-Dibromopropane (1,3- DiBP)
8r
1-Bromo- 3-chloropropane (1 -B- 3-CP)
Br
WBr W
C
I
Figure 1. Structures and abbreviations of halogenated propanes. bromo-3-chloropropane (Figure l),1,2-bis(triphenylphosphino)ethane, 2,3-dichloropropan-l-ol, 1,3-dichloropropan-2-01, 1,3-dibromopropan-2-01, bromine, dichloromethane, chloromethane, diethyl ether, and thionyl chloride from Aldrich-Chemie, Steinheim, FRG; 1,2,3-tribromopropane (Figure 1)and proteinase K from Merck, Darmstadt, F R G calf thymus DNA, trypsin (type 111-S), and collagenase (CLS 11,150 unita/mg) from Worthington Biochemical Co., Freehold, NJ; dimethyl sulfoxide (DMSO), n-heptane, pentane, and hexane from Rathburn, Walkernburn, Scotland; and Hoechst 33258 from Calbiochem-Boehringer, La Jolla, CA. Other chemicals were analytical grade from commercial suppliers. Synthesis. 1,3-Dibromc-2-chloropropane used in these studies was prepared by chlorination of the dibrominated alcohol with thionyl chloride (23).Thionyl chloride (13.0 g, 0.11 mol) was added dropwise to a solution of 1,3-dibromopropan-2-01(21.7g, 0.10 mol) in CH2C12(100 mL). The solution was stirred at room temperature for 16 h followed by 30 min of refluxing. The solution was then diluted with hexane (200 mL), extracted with saturated aqueous sodium bicarbonate (2 X 300 mL), extracted with H 2 0 (2 X 300 mL), and dried over sodium sulfate. The solution was filtered, the filtrate was evaporated, and the resulting oil was vacuum distilled (88-92 "C at 5 mm Hg) to yield a colorless oil. The fiial product was 94% pure by GC analysis: 'H NMR (CDCI,, 300 MHz) 6 4.70 (1 H, m; C-2H) and 4.22 (4 H, m; C-1 and C-3 methylene); EI-MS, m/z 203/201/199 ( M + - Cl), 159/157/155 (Me+- Br), 145/143/141 (M'+ - CH2Br). l-Bromo-2,3-dichloropropane was synthesized by bromination of the corresponding dichloro alcohol using the procedure described by Schmidt and Brooks (24). Liquid bromine (10 mmol) in CHzC12(10 mL) was added dropwise to a solution of 1,2-bis(triphenyl-1-phosphino)ethane (10 mmol) in CHzC12(50 mL) at 0 "C. Following the complete addition of bromine, a solution of 2,3-dichloropropanol(8 mmol) in CH2C12(10 mL) was added at a rate that maintained the temperature a t 0 OC. The resulting solution was allowed to warm to room temperature and stirred for 12 h. After 12 h, 400 mL of a pentane/diethyl ether (21) solution was added and the mixture filtered through a thin pad of silica gel. The filtrate was evaporated to a light yellow oil which was vacuum distilled (52-54 "C at 10 mmHg) to yield 1.1g of a colorless oil (7.3 mmo1,73% yield). The final product was >95% pure by GC analysis: 'H NMR (CDCI,, 300 MH,) 6 4.28 (1 H, m; C-2H), 3.92 (2 H, m; C-3 methylene), and 3.77 (2 H, m; C-1 methylene); EI-MS, m / z 115/113/111 (M+ - Br), 77/75 (M+ Br - HC1).
LAg et al.
530 Chem. Res. Toxicol., Vol. 4, No. 5, 1991 Table 1. Kidney Toxicity Induced by Halogenated Propanes'
kidney necrosis
treatment control, DMSO 1,2,3-tribromopropane (1,2,3-triBP)
1,2-dibromo-3-chloropropane (1,2-diB-3-CP,DBCP)
1,2,3-trichloropropane (1,2,3-triCP)
1,2-dibromopropane (1,a-diBP)
1,3-dibromopropane (l,&diBP)
1-bromo-3-chloropropane(l-B-3-CP)
85 170 85 170 170 340 510 500 750 1000 500 750 1000 1000 2000 3000 750 1000 1500 500 750 1000 1000 2000 3000
kidney/ body wt X 102 0.74 f 0.06 0.92 f 0.04 1.00 f 0.07 0.90 f 0.14 0.95 f 0.11 0.75 f 0.04 0.88 f 0.09 0.93 f 0.15 0.87 f 0.11 0.92 f 0.13 0.97 f 0.04 0.80 f 0.07 0.81 f 0.02 0.86 f 0.03 0.81 f 0.06 0.92 f 0.16 1.04 0.75 f 0.03 0.81 f 0.09 0.80 f 0.08 0.75 f 0.06 0.79 f 0.04 0.98 f 0.25 0.71 f 0.07 0.74 f 0.06 0.76 f 0.06
creatinine, pmol/L 61.3 f 14.1 105.4 f 37.1 268.0 f 136.1 58.0 f 20.0 175.3 f 77.9 40.6 f 10.2 198.0 f 135.0 455.0 f 54.3 66.6 f 15.2 124.0 f 96.0 244.5 f 88.4 42.8 f 6.5 53.4 f 7.8 185.6 f 101.6 67.8 f 4.5 63.3 f 2.6 79.5 73.0 f 7.6 51.8 f 8.5 114.8 f 62.1 73.2 f 5.8 77.2 f 7.6 89.0 f 22.5 73.2 f 5.8 77.2 f 7.6 89.0 f 22.5
urea, mmol/L 6.5 f 0.7 10.5 f 6.0 38.9 f 22.3 7.0f 5.6 24.7 f 14.7 3.5 f 0.6 30.9 f 22.2 84.0 f 16.0 6.1 f 2.7 24.8 f 28.2 57.2 f 19.3 5.4 f 0.6 7.6 f 4.6 54.9 f 18.4 5.4 f 1.5 9.3 f 4.5 15.9 5.5 f 0.8 7.0 f 3.4 10.3 f 4.2 5.5 f 1.3 6.8 f 2.5 15.4 f 16.6 5.5 f 1.3 6.8 f 2.5 15.4 f 16.6
0 5 0 0 2 0 5 1 0 0 0 0 3 0 0 5 4 1 5 2 1 5 5 1 5 5 5
1+ 0 1 0 2 0 0 0 0 4 3 0 2 5 1 0 0 0 0 3 2 0 0 1 0 0 0
mean gradeb 2+ 3+ 4+ f SD 0 0 0 0.0 f 0.0 1 3 0 2.4 f 0.9 0 3 2 3.4 f 0.5 1 0 0 0.8 f 0.8 1 4 0 2.8 f 0.4 0 0 0' 0.0 f 0.0 1 3 0 2.2 f 1.3 0 0 4 4.0 f 0.0' 1 0 0 1.2 f 0.4 0 2 0 1.8 f 1.1 1 2 2 3.2 f 0.8 0 0 0 0.4 f 0.5 0 0 0 1.0 f 0.0 0 2 2 3.0 f 1.2 0 0 0 0.0 f 0.0 0 0 0 0.0 f 0.0' 1 0 0 1.0c 0 0 0 0.0 f 0.0 0 0 0 0.6 f 0.5 2 0 0 1.2 f 0.8 0 0 0 0.0 f 0.0 0 0 0 0.0 f 0.0 1 0 0 1.0 f 1.w 0 0 0 0.0 f 0.0 0 0 0 0.0 0.0 0 0 0 0.0 f 0.0
*
'Five animals in each treatment group were dosed ip with one of the halogenated propanes. All animals were killed 48 h after administration of the test compound. bCalculated by adding together the necrosis grade for each animal divided by the total number of animals. Deaths were noted in this treatment group. elution profile approaches a straight line, NAAC is reduced to the first-order logarithmic elution rate. In Vivo Testicular DNA Damage. Testicular DNA damage was determined by the same alkaline elution system as used for renal DNA damage. The animals were killed 120 min after the ip administration of the test substance, and their testes were removed. A mixed population of testicular cells and nuclei was prepared according to a similar method as described for the kidney nuclei (26, 29). In short, the testes were removed and placed quickly in ice-cold Merchant's solution, decapsulated, and strained through a steel screen (0.4 mm). The resulting crude preparation was filtered and centrifuged two times (200g for 4 min). The final preparations consisted mostly of primary and secondary spermatocytes, and less than 5% sperm cells were observed by microscopic examination of stained cells. Samples were loaded onto polycarbonate filters, and DNA was eluted and determined as described above. In Vitro Testicular DNA Damage. Testicular cells were prepared with testes pooled from 3 animals. The testes were decapsulated, and seminiferous tubules were incubated a t 33 "C with collagenase and trypsin, as previously described (12,30).To remove large fragments and spermatozoa, the suspensions were filtered through gauze and a nylon mesh and centrifuged three times. Cellular viability was greater than 95% as measured by trypan blue exclusion. Suspensions of testicular cells (2 X 1@/mL, mostly primary and secondary spermatocytes) in 2 mL of Hank's-Hepes buffer with 1% albumin were exposed to DBCP (1 and 2.5 pM), 1,2,3-tribromopropane (1 and 2.5 pM), 1,3-dibromo-2-chloropropane (2.5, 5 , and 10 pM), 1,3-dichloro-2bromopropane (10,50, and 100 pM), l-bromo-2,3-dichloropropane (10, 50, and 100 pM), or 1,2-dibromopropane (50 and 100 pM) a t 33 "C for 60 min. The propane analogues were dissolved in DMSO, and the final concentration of DMSO in the incubations was 0.25% v/v of the medium (DMSO alone served as the control). After incubation, the cells were assayed for DNA damage by the automated alkaline elution system (26). Tissue a n d Plasma Concentrations. Kidney, testis, and
plasma concentrations of the halogenated propanes were determined according to a modified version of the method of Ruddick and Newsome (31). Animals, 4 rata per treatment group, were killed 1, 3, or 8 h after ip administration of 85, 340, 500, 1O00, or 1300 pmol/kg of the respective propanes. The selection of the dose was dependent on the toxicity of the compound. Blood samples were collected in heparinized tubes, and kidneys and testes were immediately removed, weighed, and placed in 10% (w/v) of cold absolute ethanol. Appropriate amounts of DBCP or 1,2-dibromo-4-chlorobutanewere added as intemal standards. Further preparation of the blood and tissue samples was performed as described earlier (11). Finally, the samples were extracted with heptane, and 2 p L of the heptane phase was injected into a Perkin-Elmer (Model 8700) GC. The GC unit was equipped with a B3Nielectron capture detector and a 12 m X 0.22 mm (i.d.) fused silica capillary column coated with BP-1 as stationary phase (SGE,Australia). Analysis of the halogenated propanes was performed under the following conditions: carrier gas He (head pressure, 6.6 psi); injector temperature 250 "C; detector temperature 300 "C; temperature program splitless injection a t oven temperature of 60 OC, then raised after 2 min by 10 "C/min to 140 OC followed by 30 OC/min to 260 "C.
Results Nephrotoxicity of Halogenated Propanes. Nephrotoxicity of halogenated propanes was determined morphologically and by clinical measurements 48 h after administration of the test compounds. Extensive kidney necrosis and increased kidney t o body weight ratios were evident at low doses of both DBCP and 1,2,3-tribromopropane (85 kmol/kg) (Table I). 1,2,3-Tribromopropane appeared to be somewhat more nephrotoxic than DBCP. Higher doses of both compounds (170 pmol/kg) were however required to induce significant increases of plasma creatinine and urea concentrations. 1.3-Dibromo-2-
Organ Toxicity of Halogenated Propanes 0
Chem. Res. Toxicol., Vol. 4, No. 5, 1991 531 o 1,2,3-TriBP 0 DBCP (1.2-DiB-3-CP) 0 1,3-DiB-2-CP 1-9-2.3-DiCP A 1,3-DiC-2-BP
DBCP (1,2-DiB-J-CP) 1 -B-2,3-DiCP
A 1.3-DiC-2-BP
--
1.2-DiBP
140
‘L
c
0
120
I
0,
100
Y
0
80
Z
60
a a
10
100
1000
10000
pmol/kg
40 20 0 1
10
100
1000
10000
pmol/kg Figure 2. In vivo renal DNA damage, measured by alkaline elution, 60 min after various ip doses of 1,2,3-tribromopropane (1,2,3-triBP),1,2-dibromo-3-chloropropane(1,2-diB-3-CP,DBCP), 1,3-dibromo-2-chloropropane(1,3-diB-2-CP), 1,3-dichloro-2bromopropane (1,3-diC-2-BP),l-bromo-2,3-dichloropropane (1B-2,3-diCP), 1,2,3-trichloropropane (1,2,3-triCP), 1,2-dibromopropane (1,2-diBP), 1,3-dibromopropane (1,3-diBP), or 1bromo-3-chloropropane (1-B-3-CP). The data are expressed as normalized area above curve (NAAC; see Materials and Methods). NAAC values in the dotted area are not different from the control value (7 f 2). Values are means f SD from three individual rats.
chloropropane, which unlike DBCP has the two bromines in nonvicinal positions, was less nephrotoxic. Extensive kidney necrosis, increased plasma creatinine, and increased urea levels were observed at 340 pmol/ kg 1,3-dibromo-2chloropropane. By substituting one bromine with a chlorine as in 1,3-dichloro-2-bromopropane and l-bromo2,3-dichloropropane, the nephrotoxicity was clearly decreased, since kidney necrosis was first observed at 500 pmol/ kg. Further, 1,2,3-trichloropropaneshowed very low potency as a nephrotoxicant. Of the dihalogenated propanes, 1,2-dibromopropane and 1,3-dibromopropanewere approximately 10 times less toxic than DBCP, whereas 1-bromo-3-chloropropanedid not cause any detectable kidney toxicity, even at 3000 pmol/kg (Table I). In Vivo Renal DNA Damage. Renal DNA damage, as determined by alkaline elution of DNA from isolated nuclei, was studied 60 min after ip injection of the halogenated propanes in rats. DBCP and 1,2,3-tribromopropane induced renal DNA damage at a very low dose (5 pmol/ kg) (Figure 2), whereas 1,3-dibromo-2-chloropropane caused substantial DNA damage at 20 pmol/kg and higher. Doses >25 pmol/kg were required to induce and 1DNA damage by 1,3-dichloro-2-bromopropane bromo-2,3-dichloropropane. The analogue with three chlorines, 1,2,3-trichloropropane,was even less potent and caused significant DNA damage only at doses 1375 pmol/ kg. Of the dihalogenated compounds, 1,2-dibromopropane also caused renal DNA damage at 375 pmol/ kg, whereas neither 1,3-dibromopropane nor 1bromo-3-chloropropanecaused DNA damage at the highest dose tested (3000 pmol/kg) (Figure 2). Testicular Toxicity. The testicular toxicity was determined histologically in rats 10 days after administration of the test compounds. Testicular necrosis and atrophy of seminiferous tubules were observed after a single ip dose of 170 pmol/ kg DBCP and 1,2,3-tribromopropane. 1,3-
Figure 3. In vivo testicular DNA damage, measured by alkaline elution, 120 min after ip doses of 1,2,3-tribromopropane (1,2,3triBP), 1,2-dibromo-3-chloropropane(1,2-diB-3-CP, DBCP), 1,3-dibromo-2-chloropropane(1,3-diB-2-CP), 1,3-dichloro-2bromopropane (1,3-diC-2-BP), l-bromo-2,3-dichloropropane (1B-2,3-diCP), or 1,Zdibromopropane (1,2-diBP). The data are expressed as normalized area above curve (NAAC; see Materials and Methods). NAAC values in the dotted area are not different from the control value (15 f 1). Values are means f SD from three individual rats.
Dibromo-2-chloropropanewas less potent, and a dose of 340 pmol/ kg was needed to cause testicular damage, while 1000 pmol/kg was required to cause testicular injury by 1,3-dichloro-2-bromopropane.The damage was only observed in one animal, and two of the five animals in this group died. Doses up to 1000 pmol/kg did not cause any testicular injury by l-bromo-2,3-dichloropropaneor 1,2dibromopropane; however, deaths were noted at the highest doses tested. The other analogues used in the nephrotoxicity study (1,3-dibromopropane, l-bromo-3chloropropane, and 1,2,3-trichloropropane)were not tested for testicular toxicity, because of their presumed low toxic potential. In Vivo Testicular DNA Damage. Testicular DNA damage was determined by alkaline elution 120 min after ip injection of the halogenated propanes in rats. DBCP and 1,2,3-tribromopropaneinduced testicular DNA damage at 85 pmol/kg (Figure 3). A 170 pmol/kg concentration of 1,3-dibromo-2-chloropropanewas necessary to get a similar degree of damage as 85 pmol/kg DBCP. Of the two dichlorobromo compounds, l-bromo-2,3-dichloropropane was the most potent in causing testicular DNA damage, since a dose of 170 pmol/kg l-bromo-2,3dichloropropane induced an equivalent degree of DNA damage as 500 pmol/ kg 1,3-dichloro-2-bromopropane. However, both of these compounds caused less DNA damage than did 1,3-dibromo-2-chloropropane.1,2-Dibromopropane did not cause any DNA damage even at 2000 pmollkg (Figure 3). In Vitro Testicular DNA Damage. To study cellular activation of the different halogenated propanes to DNAdamaging intermediate(s), cells isolated from testes of rats were incubated together with the propanes for 60 min. The DNA-damaging effects were assessed in the isolated cells according to the alkaline elution method. At 1p M concentrations, both DBCP and 1,2,3-tribromopropanecaused testicular DNA damage, with 1,2,3-tribromopropaneas the most potent analogue (Figure 4). 1,3-Dibromo-2-chloropropane at 2.5 p M induced a similar extent of DNA damage as did 1pM DBCP. Higher concentrations (10-100 p M ) were required of 1,3-dichloro-2-bromopropaneand l-bromo-2,3-dichloropropaneto cause DNA damage, whereas no appreciable effect on alkaline DNA elution was
LAg et al.
532 Chem. Res. Toxicol., Vol. 4,No.5, 1991
Table 11. Testicular Necrosis and Atrophy Induced by Halogenated Propane@ testicular injury dose, testis/body mean treatment pmol/kg wt X lo2 0 1+ 2+ 3+ 4+ gradeb* SD control, DMSO 1.12 f 0.05 5 0 0 0 0 0.0 i 0.0 1,2,3-tribromopropane (1,2,3-triBP) 0 0 0 0.0 f 0.0 85 1.22 f 0.07 5 0 1 0 2 2.0 f 2.0 2 0 170 1.12 f 0.14 1,2-dibromo-3-chloropropane (1,2-diB-3-CP, DBCP) 85 1.29 f 0.06 0 0 0 0.0 f 0.0 5 0 1 0 3 2.7 f 2.3 0 1 170 0.96 f 0.16 1,3-dibromo-2-chloropropane(1,3-diB-2-CP) 170 1.26 f 0.09 0 0 0 0.0 f 0.0 5 0 0 0 1 1.7 f 2.1' 1 1 340 1.20 f 0.14 1,3-dichloro-2-bromopropane (1,3-diC-2-BP) 500 1.21 f 0.18 5 0 0 0 0 0.0 f 0.0 0 0 1 1.3 f 2.3' lo00 1.01 f 0.45 2 0 l-bromo-2,3-dichloropropane(l-B-2,3-diCP) 500 5 0 0 0 0 0.0 f 0.0 1.11 f 0.08 0 0 0 0.0 f 0.0' 3 0 lo00 1.10 f 0.13 1,2-dibromopropane (1,2-diBP) 500 1.15 f 0.08 5 0 0 0 0 0.0 f 0.0 lo00 1.22 2 0 0 0 0 0.0' "Five animals in each treatment group were dosed ip with one of the halogenated propanes. All animals were killed 10 days after administration of the test compound. Calculated by adding together the necrosis grade for each animal divided by the total number animals. 'Deaths were noted in this treatment group. 240 220
.
4
I
o 1.2.3-TriBP 0 DBCP (1.2-DiB-3-CP) 0 1.3-DiB-2-CP 1-B-2,3-DiCP A 1.3-DiC-2-BP + 1,2-DiBP
160
I
20 0
1
10
100
1000
PM
Figure 4. In vitro testicular DNA damage measured by alkaline elution after exposure of testicular cells for 60 min to 1,2,3-tribromopropane (1,2,3-triBP), 1,2-dibromo-&chloropropane(1,2diB-3-CP, DBCP), 1,3-dibromo-2-chloropropane(1,3-diB-2-CP), 1,3-dichloro-2-bromopropane(1,3-diC-2-BP), l-bromo-2,3-dichloropropane (l-B-2,3-diCP), or l,2-dibromopropane (1,2-diBP). The data are expressed as normalized area above curve (NAAC; see Materials and Methods). NAAC values in the dotted area are not different from the control value (16 f 3). Values are means f SD from three separate experiments.
observed even at 100 p M 1,2-dibromopropane(Figure 4). Plasma and Tissue Concentrations. The differences in the extents of renal and testicular injury and DNA damage caused by the halogenated propanes could be due to different tissue concentrations of the propanes, and this possibility was further investigated. Depending on their toxicity, the compounds were administrated at different dose levels. The plasma and tissue concentrations of the halogenated propanes were determined in animals 1,3, and 8 h after a single ip administration. The renal and testicular concentrations were higher than the plasma concentrations at all time points (Figure 5). Thus, all of the halogenated propanes were, as reported previously for DBCP (11, 13), accumulated in the kidney and testis. 1,2,3-Tribromopropane at 85 pmol/ kg appeared to be eliminated faster from the plasma, kidney, and testis than DBCP (Figure 5 ) . Further, the plasma elimination of 1,2,3-trichloropropanewas substantially slower than that of 1,2,3-tribromopropane and DBCP. The halogenated propanes, which were less toxic and were administrated at higher dose levels (340,500,1000, and 1300 pmol/kg)
Table 111. Relative Organ-Injurying and DNA-Damaging Potencies of Some Selected Halogenated Propanes relative potency in vivo DNA in vitro DNA organ injury damageb damage' substance kidney testis kidney testis testis 64 1,2,3-tribromo+++ +++ 4.7 0.6 propane 1,2-dibromo-3+++ +++ 4.7 0.5 36 chloropropane 1,3-dibromo-2++ ++ 1.3 0.3 15 chloropropane 1,3-dichlor0-2++ + 0.4 0.04 0.8 bromopropane 0.5 0.08 1.2 l-bromo-2,3-di++ 0 chloropropane 1,2-dibromo+ o 0.06 0 0.3 propane a(+++) lgrade 2.0 injury at doses up to 170 pmol/kg. (++) lgrade 1.0 injury at doses up to 750 pmol/kg. (+) lgrade 1.0 injury at doses up to 1500 pmollkg. bThe NAAC to dose ratio. The dose in the middle of the administered dose range has been used. 'The NAAC to concentration ratio. The concentration in the middle of the applied concentration range has been used.
than DBCP and 1,2,3-tribromopropane,also showed higher time-dependent tissue concentrations.
Discussion A number of halogenated propanes were studied for renal and testicular necrogenic effects in the rat and correlated with their ability to induce renal and testicular DNA damage in vivo and also testicular DNA damage in vitro. A considerable variation in the extents of target organ toxicity (i.e., organ necrosis and DNA damage) between the halogenated propanes was observed. The halogenated propanes showed a similar rank order of necrosis in kidney and testis. DBCP and 1,2,3-tribromopropane were the most potent of the propanes tested that caused tissue damage in both kidney and testis. However, higher doses of the propanes were required to induce testicular injury than kidney injury (Tables I and 11). Further, 1,3-dibromo-2-chloropropane was less toxic than DBCP and 1,2,3-tribromopropane in these two organs, but more toxic than the dichloromonobrominated analogues. l-Bromo-2,3-dichloropropaneand 1,2-dibromopropane induced kidney necrosis but caused no toxic
Organ Toxicity of Halogenated Propanes
Chem. Res. Toxicol., Vol. 4, No. 5, 1991 533
0
L1 .-c v)
2 1000
,
10000
1000
500 p n o l l k g 85 umollkg
1000 gmollk!
340 j ” l / 1000
100
10
~~
1
1.2,3-TriBP
3h 8h DBCP
t, E 1,3-DiB-Z-CP
l h 3h 8 h 1,3-DiC-Z-BP
1 -8-2.3-DiCP
h 8h 1 ,P-DiBP
’
l h 3h 8 h 1,3-DiBP
l h 3h 8h 1,2,3-TriCP
l h 3h 8h 1-8-3-CP
E Figure 5. Plasma, kidney, and testicular concentrations of various halogenated propanes after a single dose of either 85 pmol/kg (1,2-diB-3-CP,DBCP); 340 pmol/kg 1,3-dibromo-2-chloropropane 1,2,3-tribromopropane(1,2,3-triBP)and 1,2-dibromo-3-chloropropane (1,3-diB-2-CP);500 pmol/kg 1,3-dichloro-2-bromopropane(1,3-diC-2-BP)and l-bromo-2,3-dichloropropane(l-B-2,3-diCP);lo00 pmol kg 1,2-dibromopropane(1,2-diBP)and 1,3-dibromopropane(1,3-diBP);or 1300 pmollkg 1,2,3-trichloropropane(1,2,3-triCP)an 1bromo-3-chloropropane (l-B-3-CP). Values are means f SD from four individual rats.
d
effects in testis at the highest dose administered. Higher doses could not be used because of their lethal effects. These results on renal and testicular injury show that both the type, the number, and the position of the halogens affect organ-damaging activity (Table 111). Tissue distribution patterns of the halogenated propanes were examined to exclude the possibility that this could be the reason for the differences in their capacity to induce toxicity. Administration of DBCP and 1,2,3-tribromopropane, at doses that caused organ damage, resulted in lower renal and testicular concentrations than those of the other halogenated propanes at doses that caused little or no damage. Thus, differences in tissue concentrations cannot explain the observed variations in toxic potential between the various propanes. The ability of the halogenated propanes to cause renal DNA damage correlated well with their ability to induce kidney damage (Table HI), with the exception of 1,3-dibromopropane. This compound did not induce DNA damage, although it caused kidney necrosis at high doses (1000 pmol/kg). This may indicate that the nephrotoxic mechanism of 1,3-dibromopropane may be different from that of the other halogenated propanes. Also in testis there was a good correlation between necrosis and in vivo DNA damage (Table 111). The only exception was l-bromo2,3-dichloropropane, which induced testicular DNA damage without causing any observable testicular necrosis or atrophy at the highest dose tested. Higher doses could not be used because of its general toxicity. Further, in vivo testicular DNA damage of the halogenated propanes correlated well with the in vitro DNA damage observed in isolated testicular cells (Table 111). These latter observations indicate that the reactive metabolite inducing DNA damage can be generated in the testicular cells in situ for all of the halogenated propanes, as demonstrated previously for DBCP (12).In analogy with DBCP (11,13, 14), the other propanes also induced DNA damage at shorter times after exposure and at lower doses than those causing organ necrosis. The dose and time relationships for DNA damage, as well as the good correlation between DNA damage and organ damage of the different propanes,
further substantiate the proposal that DNA damage may be an initial event in the development of organ necrosis caused by halogenated propanes. The variation in toxicity among the halogenated propanes may be due to factors such as differences in their rate of metabolism to reactive intermediates, differences in the reactivity of the intermediates with target and nontarget molecules, or differences in the rate of repair of damage caused by the reactive intermediates. The present results show that the type, the number, and the positions of the halogens affect the toxicity of the test compounds (Table 111). In accordance with bromine being a better leaving group than chlorine, 1,2,3-tribromopropane was found to be much more potent than 1,2,3-trichloropropane and the dibromomonochlorinated compounds were more toxic than the dichloromonobrominated analogues. This might be related to differences in their formation of glutathione conjugates. The rate of formation of S-propylglutathione is earlier reported to be faster from propyl bromide than propyl chloride (4). Halogen substituents at all three carbons are also essential for toxic activity, since DBCP and 1,3-dibromo-2-chloropropane were much more toxic than dibrominated propanes lacking the third halogen (1,2-dibromopropane and 1,3-dibromopropane). In addition, the presence of two bromines in vicinal positions appears to be of importance, since DBCP was more toxic than 1,3-dibromo-2-chloropropane. The availability of a good leaving group in the C2-position presumably promotes formation of reactive episulfonium ions from monoglutathione conjugates (8). These structure-activity relationships of halogenated propanes with respect to tissue injury and DNA damage are in agreement with earlier reported results from dominant lethal studies in male rats where the halogenated compounds DBCP, 1,2,3-tribromopropane, 1,2-dibromopropane, 1,2,3-trichloropropane, bromopropane, and 1-chloropropane were used (32). In conclusion, the present study has established a good relationship between in vivo organ necrogenic effects and DNA damage for a series of halogenated propanes. Both the type, the number, and the position of the halogens
534 Chem. Res. Toxicol., Vol. 4, No. 5, 1991
affected the toxic potential. The most toxic of the halogenated propanes contain three halogens with at least two vicinal bromines.
Acknowledgment. These studies were supported by NIH Grant ES02728 and research grants from the Norwegian Council for Science and Humanities and the Royal Norwegian Council for Scientific and Industrial Research. We appreciate very much the technical assistance of Bente Trygg and Kirsti Bekkedal and the typing performed by Anne Lene Solbakken. Registry No. 1,2,3-TriBP, 96-11-7; DBCP, 96-12-8; 1,3-DiB3-CP, 51483-40-0; 1,3-DiC-2-BP, 51483-39-7; l-B-2,3-DiCP, 33037-07-9; 1,2,3-TriCP, 96-18-4; 1,2-DiBP, 78-75-1; 1,3-DiBP, 109-64-8; 1-B-3-CP, 109-70-6; 1,3-dibromopropan-2-01, 96-21-9; 2,3-dichloropropanol, 616-23-9.
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