Strong nasal carcinogenicity and genotoxicity of 1-nitroso-4-methylpiperazine after low dose inhalation in rats
R.G. Klein,
P. Schmezer1,
R. Hermann,
P. Waas,
B. Spiegelhalder and
H. Bartsch
Deutsches Krebsforschungszentrum, Division of Toxicology and Cancer Risk Factors, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
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Abstract
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SpragueDawley rats were exposed by inhalation to 1-nitroso-4-methylpiperazine (NMPz) vapor at 2.4 p.p.m. for 15 h/day for 74 days over a 7.5 month period. After a dose of 1.1 mg/day NMPz (total dose 340 mg/kg body wt) 10/10 animals developed tumors of the nasal cavity, mostly invasive muco-epidermoidal carcinomas; no such tumors were observed in sham-exposed controls. This high tumor yield was seen at an 80 times lower dose and a shorter latency period when compared with rat carcinogenicity studies reported earlier. The single cell microgel electrophoresis (Comet) assay was used to determine genotoxicity in target tissues. Short-term in vitro exposure of rat and human nasal epithelial tissues to NMPz caused genotoxic effects in cells of both species. Short-term in vivo exposure of rats to NMPz vapor for 1 h induced DNA damage in nasal epithelial cells. Our results revealed NMPz as a potent genotoxic nitrosamine in rat and human nasal cells, the carcinogenicity of inhaled NMPz vapor in rats being remarkably higher as compared with oral uptake.
Abbreviations: EMEM, Eagle's minimum essential medium; NDMA, N-nitrosodimethylamine; NMPz, 1-nitroso-4-methylpiperazine; TEA, thermal energy analyzer.
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Introduction
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The formation of volatile carcinogenic N-nitrosamines from secondary amines can pose hazards to workers exposed by inhalation, especially in the rubber industry (1). In order to prevent such nitrosamine exposure one approach aimed to develop `safe amines' that upon nitrosation would yield non-carcinogenic nitrosamines. One such promising amine was 4-methylpiperazine, which could be used as a vulcanization accelerator in the rubber industry (2). At that time published bioassay data (37) on the corresponding nitrosamine, 1-methyl-4-nitrosopiperazine (NMPz), were only available after oral application of a total dose of 10 and 26 g/kg body wt, respectively. In two studies in rats NMPz showed a carcinogenic effect in the nasal region only after very high dosages (57), but it was evaluated as negative in two rodent bioassays (3,4). However, NMPz was reported to be mutagenic in several short-term tests (816). Because workers in the rubber industry could be potentially exposed to NMPz vapor we have tested the carcinogenicity of NMPz in a pilot inhalation study. Its genotoxicity after short-term in vitro and in vivo exposure of rat and human nasal tissue was assessed by using the single cell microgel electrophoresis (Comet) assay. We here report the unexpected high sensitivity of nasal epithelial cells of rodent and human origin to the genotoxic and carcinogenic action of NMPz.
NMPz was synthesized according to Preussmann et al. (17) and a purity of 99.5% was established by GC-MS and thermal energy analyzer (TEA) analysis.
Seventeen 2-month-old female SpragueDawley rats (Charles River/Wiga, Hannover, Germany) were randomly distributed into two groups, consisting of 10 treated animals and seven sham-exposed controls. This rat strain is sensitive to induced nasal carcinogenicity by N-nitrosodimethylamine (NDMA) after inhalation (18). Rats were housed in Macrolon type III cages and fed a standard diet during recreation time and tap water. Before exposure to NMPz vapor, two animals were placed in stainless steel wire cages without food, but with drinking water, inside the inhalation chamber (500 l volume, air exchange 6 times/h). The position of the wire cages in the boxes was changed continuously for each new exposure day in order to average exposure concentration and flow differences caused by chamber geometry.
NMPz vapor was generated by evaporation at room temperature in a glass vessel of 200 ml volume, using a flow meter to regulate the auxiliary air stream, and then mixed with the main air stream controlled by a second meter. For safety reasons the system was kept under negative pressure. Exposure to NMPz vapor was done for 14.5 h/day from 5 p.m. to 7.30 a.m. As rats were more active overnight they were expected to inhale a higher dose of the carcinogen to be tested. After exposure the rats were transferred from the wire cages into Macrolon housing cages in a separate chamber provided with food and water. The NMPz concentration inside the exposure chambers was analyzed continuously by Impinger absorption in distilled water and UV detection at 230 nm using calibration curves; measurements were further confirmed by parallel Thermosorb tube absorption and TEA analysis (18). The exposure was terminated after 74 days inhalation over 7.5 months. The mean breathing volume of the animals during NMPz exposure was calculated from the measured CO2 concentration of the chamber air and the air flow in the chamber. Thereby it was assumed that the expired air from the rats had a physiological CO2 content of 4%. The sham-exposed controls were kept in wire cages without food, but with drinking water, for the same time in an identical inhalation box without NMPz exposure.
Treated animals were killed by CO2 in the air stream when moribund, control rats were killed 350 days after the start of inhalation exposure. Organs were fixed in 10% neutralized formaldehyde. Histopathological sections were stained with hematoxylin and eosin and examined by Drs D.Komitowski and F.Amelung (Division of Histodiagnostics and Pathomorphology, Deutsches Krebsforschungszentrum, Heidelberg, Germany).
Human nasal epithelium biopsies were obtained from male healthy donors aged 2260 years undergoing surgery for removal of obstructions (Prof. H.Maier, ENT Hospital, University of Heidelberg, Heidelberg, Germany). Rat nasal epithelium was excised from treated or control F344 rats (Wiga, Sulzfeld, Germany) immediately after death. Tissue samples of both species were incubated in a shaking water bath at 37°C for 30 min, in Joklik's modified Eagle's minimum essential medium (EMEM) (Linaris, Bettingen, Germany) supplemented with 46 U/ml pronase E (Sigma-Aldrich, Deisenhofen, Germany), collagenase P (100 U/ml, with an additional trypsin activity of ~0.8 U/ml) and hyaluronidase (1000 U/ml) Böhringer, Mannheim, Germany). The resulting cell suspension was washed with enzyme-free EMEM, centrifuged and adjusted to 1x106 cells/ml. In the case of the in vitro test, the isolated mucosa cells were incubated at different NMPZ concentrations for 1 h in a shaking water bath at 37°C and subsequently subjected to an alkaline version of the single cell microgel electrophoresis (Comet) assay as described elsewhere (19,20). For the in vivo studies, the mucosa cells were prepared immediately after the inhalation exposure (5 mg/kg body wt NMPz for 1 h) and subjected directly to Comet analysis. In this pilot study no attempts were made to separate different cell types. Cell viability was determined by Trypan blue exclusion before the cells were analyzed for DNA damage. The Comet assay was used to detect the induction of DNA damage. The overall comet length (in µm) of individual DNA spots from 50 cells/slide and 13 slides/data point were measured.
A summary of exposure data and results on tumor yield in treated SpragueDawley rats and controls after NMPz inhalation are given in Table I
. As NDMA exposure is known to occur in the rubber industry we have included for comparative purposes its carcinogenic response in the same rat strain after inhalation (Table I
). Differences in body weight gain between NMPz-treated and control rats were negligible before tumor appearance. The first tumor was observed after 205 days and the last after 330 days survival (median latency period 301 days). All 10 treated rats developed tumors in the nasal region after an estimated dose of 1.1 mg/rat/day NMPz, corresponding to an uptake of 85110 mg/rat NMPz (340 mg/kg body wt NMPz). Except for one adenocarcinoma and one olfactory neuroblastoma all other tumors were multifocal muco-epidermoid carcinomas. Most of them invaded the basal skull and the frontal brain.
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Table I. Inhalation studies (whole body exposure) with NMPz (15 h/day overnight) and comparison with NDMA (4 h/day, 4 days/week; taken from ref. 18)
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Control rats were killed after 350 days, immediately after the histopathological confirmation that the last treated rat had developed a nasal tumor. No other significant non-neoplastic histopathological changes were observed in the nasal tissue of the treated rats. Results on genotoxicity after short-term in vivo and in vitro exposure of rat and human nasal tissues to NMPZ are summarized in Table II
. After 1 h incubation in the presence of 12.550 mM NMPz a significant increase in overall comet length in cells of both species was observed, indicative of single-strand breaks and alkali-labile sites as primary induced DNA lesions. No cytotoxic effect was found by Trypan blue exclusion within the concentration range tested. The inter-individual differences were somewhat higher in human than in rat samples, leading to different levels of statistical significance, nevertheless, a similar sensitivity to the genotoxic activity of NMPZ was observed for both species. After short-term inhalation exposure of rats to NMPZ (Table III
, mean total uptake of NMPZ 5 mg/kg body wt) a significant genotoxic effect in nasal mucosa cells of the animals was induced. The viability of the isolated cells was higher in comparison with the above in vitro studies, probably because the isolated cells were subjected to Comet analysis immediately after isolation from the excised tissue, with no additional incubation step.
Data from four rodent bioassays reported conflicting results on the carcinogenicity of NMPz: In a study by Druckrey et al. (3) 16 rats received 25 mg/kg body wt NMPz daily in the drinking water, 7 days/week for life. Although malignomas were found in four rats (one carcinoma of the liver, two carcinomas of the nasal region and one sarcoma of the lower jaw), the authors evaluated NMPz as non-carcinogenic. Lijinsky and Taylor. (4) reported negative results in 15 (male and female) SpragueDawley rats when they received a 0.025% NMPz solution in drinking water, 5 days/week for 50 weeks. At a total dose of 1250 mg/rat NMPz, lifespan was not diminished and no significantly enhanced tumor incidence was observed against controls. However, when 20 male Fischer rats received a 0.13% solution of NMPz in the drinking water (corresponding to 26 mg/rat/day), 5 days/week until death, all rats died within 1 year because of nasal tumors (6). Reznik-Schüller treated 52 male F344 rats with 26 mg/rat/day in the drinking water (0.13% solution, 5 days/week) and examined the nasal region histopathologically up to a maximum of 40 weeks of treatment. Nasal tumors were found in all animals (7).
We have observed a 100% tumor yield at a 7080 times lower total dose of NMPz, i.e. 86 mg/rat in our inhalation studies with female SpragueDawley rats compared with 6500 mg/rat total dose orally (6,7). From these findings we conclude that inhalation exposure of rats to NMPz leads to a carcinogenic effect that is at least two orders of magnitude greater and, therefore, this route of exposure seems more adequate for evaluating possible hazards in the case of human exposure to NMPz vapor or of other volatile nitrosamines. Although metabolism of NMPz by nasal epithelial cells has not been studied in detail, it is known that human/rat nasal tissues contain cytochrome P450 isoforms (21,22), which can activate carcinogenic N-nitrosamines, such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and NDMA. This is further supported by our results in the Comet assay, which revealed that both rat and human nasal tissue is sensitive to the DNA damaging effects of NMPz.
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Notes
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1 To whom correspondence should be addressed Email: p.schmezer{at}dkfz-heidelberg.de 
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Received September 2, 1998;
revised March 31, 1999;
accepted April 20, 1999.