Organ Preservation in Rectal Cancer: An Overview of the Dutch Perspective and Recent Developments.

Although current guidelines on rectal cancer treatment often recommend rectal resection with or without neoadjuvant (chemo)radiotherapy, there is growing interest in organ-preserving treatment approaches among patients and clinicians in the Netherlands. Currently, multiple ongoing studies are investigating the value of different non-operative treatment modalities to improve tumour response rates and increase the chance of successful organ preservation. Papillon contact X-ray brachytherapy is a promising treatment modality to improve the chance of organ preservation, which seems especially relevant for elderly and frail patients unable or refusing to undergo total mesorectal excision surgery. The elderly and frail patient with rectal cancer poses a significant challenge and warrants a thorough multidisciplinary approach to provide the most optimal organ-preserving treatment. In this overview, an insight into the Dutch perspectives and developments within the field of organ preservation and the set-up of a Papillon facility to complete the spectrum of organ-preserving treatment options in a tertiary referral centre for rectal cancer treatment has been provided.

Response to radiation therapy in adrenocortical carcinoma.

INTRODUCTION: Adrenocortical carcinoma (ACC) is a rare disease which is considered resistant to many treatments. The role of radiotherapy in ACC remains unclear. In general radiotherapy is thought to be ineffective for the treatment of ACC, and therefore not often used. However, recent reports suggest the opposite. The aim of this study was to perform a retrospective analysis to evaluate the application of radiotherapy in Dutch ACC patients, and to determine the occurrence of response. MATERIALS AND METHODS: The Dutch ACC Registry (no.=159) was screened for patients who had received radiotherapy between 1990 and 2008. Tumor response evaluation was performed according to the Response Evaluation Criteria In Solid Tumors (RECIST). RESULTS: Only 13 patients (8% of registered patients) had received radiation therapy of whom 6 were irradiated for the palliation of painful bone metastases. In all patients this radiation resulted in pain relief. Three patients received adjuvant tumor bed radiation after resection. Four patients were radiated on irresectable tumor recurrence or tumor metastases. Two patients died soon after radiation therapy and therefore follow-up information regarding tumor response after radiation therapy of 2 patients was available. Interestingly, partial tumor response according to RECIST criteria, was observed in both patients. CONCLUSION: ACC can be sensitive to radiotherapy and should be considered in the treatment of advanced ACC, particularly in worrisome lesions. The role of radiotherapy in advanced ACC is to complement a systemic treatment such as mitotane or classic cytotoxic agents.

Effect of radiotherapy and chemotherapy on pulmonary function after treatment for breast cancer and lymphoma: A follow-up study.

PURPOSE: To determine the changes in pulmonary function tests (PFTs) 0 to 48 months after treatment for breast cancer and lymphoma. PATIENTS AND METHODS: The alveolar volume (V(A)), vital capacity, forced expiratory volume in 1 second, and corrected transfer factor of carbon monoxide (T(L,COc)) were measured in 69 breast cancer and 41 lymphoma patients before treatment and 3, 18, and 48 months after treatment with radiotherapy alone or radiotherapy in combination with chemotherapy (mechlorethamine, vincristine, procarbazine, prednisone, doxorubicin, bleomycin, vinblastine; cyclophosphamide, epidoxorubicin, fluorouracil; cyclophosphamide, thiotepa, carboplatin; cyclophosphamide, methotrexate, fluorouracil). The three-dimensional dose distribution in the lung of each patient was converted to the mean lung dose. Statistical analysis was used to evaluate the changes in PFT values over time in relation to age, sex, smoking, chemotherapy, and the mean lung dose. RESULTS: After an initial reduction in PFT values at 3 months, significant recovery was seen at 18 months for all patients. Thereafter, no further improvement could be demonstrated. Reductions in spirometry values and V(A) were related to the mean lung dose only (0.9% per Gy at 3 months and 0.4% per Gy mean dose at 18 months). T(L,COc) decreased 1. 1% per Gy mean dose and additionally decreased 6% when chemotherapy was given after radiotherapy. Chemotherapy administered before radiotherapy reduced baseline T(L,COc) values by 8% to 21%. All patients showed an improvement of 5% at 18 months. CONCLUSION: On the basis of the mean lung dose and the chemotherapy regimen, the changes in PFT values can be estimated before treatment within 10% of the values actually observed in 72% to 85% of our patients with healthy lungs.

Changes in local pulmonary injury up to 48 months after irradiation for lymphoma and breast cancer.

PURPOSE: To assess the recovery from early local pulmonary injury after irradiation and to determine whether regional differences exist. METHODS: For 110 patients treated for breast cancer or malignant lymphoma, single photon emission computed tomography (SPECT) perfusion and ventilation scans and CT scans were made before, 3, 18, and 48 months after radiotherapy. Dose-effect relations for changes in local perfusion, ventilation, and density were determined for each individual patient using spatially correlated SPECT and CT data sets, for each follow-up period. Average dose-effect relations for both subgroups were determined, as well as dose-effect relations for different regions. RESULTS: In general, partial improvement of local pulmonary injury was observed between 3 and 18 months for each of the three endpoints. After 18 months, no further improvement was seen. Patients with breast cancer and malignant lymphoma showed a similar improvement (except for the perfusion parameter), which was attributed to a recovery from the early radiation response and could not be explained by contraction effects of fibrosis of lung parenchyma. No regional differences in radiosensitivity 18 months after treatment were observed, except for the dorsal versus ventral region. This difference was attributed to a gravity-related effect in the measuring procedure. CONCLUSION: For all patients, a partial recovery from early local perfusion, ventilation, and density changes, was seen between 3 and 18 months after radiotherapy. After 18 months, local lung function did not further improve (lymphoma patients).

Radiation dose-effect relations and local recovery in perfusion for patients with non-small-cell lung cancer.

PURPOSE: To determine local dose-effect relations for lung perfusion and density changes due to irradiation for patients with non-small-cell lung cancer (NSCLC) and to quantify the effect of reperfusion. METHODS AND MATERIALS: For 25 NSCLC patients and a reference group of 81 patients with healthy lungs, registered single photon emission computed tomography (SPECT) lung perfusion and CT scans were made, before and after radiotherapy. Average dose-effect relations for perfusion and CT-density changes were calculated and compared with the dose-effect relation of the reference group. On the basis of these dose-effect relations, the post-RT perfusion was predicted for each patient and compared to the measured post-RT perfusion. RESULTS: Well-perfused lung regions of the NSCLC patients showed the same dose-effect relation as the reference patients. By comparing predicted and measured post-treatment perfusion scans, regions of reperfusion could be determined for 18 of 25 NSCLC patients but for none of the reference patients. CONCLUSION: Well-perfused lung tissue of patients with NSCLC behaves like healthy lung tissue with respect to radiation. The dose-effect relation for perfusion and CT density was extended for doses up to 80 Gy. Radiation damage in poorly perfused lung regions was less than predicted as a consequence of local reperfusion.

Radiation pneumonitis as a function of mean lung dose: an analysis of pooled data of 540 patients.

PURPOSE: To determine the relation between the incidence of radiation pneumonitis and the three-dimensional dose distribution in the lung. METHODS AND MATERIALS: In five institutions, the incidence of radiation pneumonitis was evaluated in 540 patients. The patients were divided into two groups: a Lung group, consisting of 399 patients with lung cancer and 1 esophagus cancer patient and a Lymph./Breast group with 78 patients treated for malignant lymphoma, 59 for breast cancer, and 3 for other tumor types. The dose per fraction varied between 1.0 and 2.7 Gy and the prescribed total dose between 20 and 92 Gy. Three-dimensional dose calculations were performed with tissue density inhomogeneity correction. The physical dose distribution was converted into the biologically equivalent dose distribution given in fractions of 2 Gy, the normalized total dose (NTD) distribution, by using the linear quadratic model with an alpha/beta ratio of 2.5 and 3.0 Gy. Dose-volume histograms (DVHs) were calculated considering both lungs as one organ and from these DVHs the mean (biological) lung dose, NTDmean, was obtained. Radiation pneumonitis was scored as a complication when the pneumonitis grade was grade 2 (steroids needed for medical treatment) or higher. For statistical analysis the conventional normal tissue complication probability (NTCP) model of Lyman (with n=1) was applied along with an institutional-dependent offset parameter to account for systematic differences in scoring patients at different institutions. RESULTS: The mean lung dose, NTDmean, ranged from 0 to 34 Gy and 73 of the 540 patients experienced pneumonitis, grade 2 or higher. In all centers, an increasing pneumonitis rate was observed with increasing NTDmean. The data were fitted to the Lyman model with NTD50=31.8 Gy and m=0.43, assuming that for all patients the same parameter values could be used. However, in the low dose range at an NTDmean between 4 and 16 Gy, the observed pneumonitis incidence in the Lung group (10%) was significantly (p=0.02) higher than in the Lymph./Breast group (1.4%). Moreover, between the Lung groups of different institutions, also significant (p=0.04) differences were present: for centers 2, 3, and 4, the pneumonitis incidence was about 13%, whereas for center 5 only 3%. Explicitly accounting for these differences by adding center-dependent offset values for the Lung group, improved the data fit significantly (p < 10(-5)) with NTD50=30.5+/-1.4 Gy and m=0.30+/-0.02 (+/-1 SE) for all patients, and an offset of 0-11% for the Lung group, depending on the center. CONCLUSIONS: The mean lung dose, NTDmean, is relatively easy to calculate, and is a useful predictor of the risk of radiation pneumonitis. The observed dose-effect relation between the NTDmean and the incidence of radiation pneumonitis, based on a large clinical data set, might be of value in dose-escalating studies for lung cancer. The validity of the obtained dose-effect relation will have to be tested in future studies, regarding the influence of confounding factors and dose distributions different from the ones in this study.

Automatic three-dimensional matching of CT-SPECT and CT-CT to localize lung damage after radiotherapy.

UNLABELLED: The aim of this study was to develop a fast and clinically robust automatic method to register SPECT and CT scans of the lungs. METHODS: CT and SPECT scans were acquired in the supine position from 20 patients with healthy lungs. After partial irradiation of the lungs by radiotherapy, the scans were repeated. Two matching methods were compared: a conventional method with external skin markers and a new method using chamfer matching of the lung contours. In the latter method, a unique value for the SPECT threshold, needed for segmentation of the SPECT lungs, was determined by iteratively applying the chamfer matching algorithm. RESULTS: The new technique for CT-SPECT matching could be implemented in a fully automatic manner and required less than 2 min. No large systematic shifts or rotations were present between the matches obtained with the marker method and the lung contour method for healthy or partially irradiated lungs. For healthy lungs, the number of ventilation SPECT counts outside the CT-defined lung was taken as a measure for a good match. This number of outside counts was slightly lower for the new method than for the conventional method, which indicates that the accuracy of the new method is at least comparable to the conventional method. For ventilation, a systematic difference between the results of the matching methods, a small translation in the anterior --> posterior direction, could be attributed to an inconsistency of the marker positions (2 mm). For perfusion, a somewhat larger anterior --> posterior shift was found, which was attributed to the gravity force. CT-CT correlation on the lung contours using chamfer matching was tested with the same dataset. For accurate matching, the CT slices encompassing the diaphragm had to be deleted. CONCLUSION: The new method based on lung contour matching is a fast, automatic procedure and allows accurate clinical follow-up.

Excretion of mutagens in human urine after passive smoking.

Eight non-smokers were experimentally exposed to cigarette smoke by staying in a poorly ventilated room together with heavy smokers for 6 h. Air samples were taken and the extract appeared to contain mutagenic substances. This is in accordance with the presence of carcinogens in tobacco smoke. Inhalation of the contaminated air by the passive-smokers resulted in an increase in the urinary excretion of products mutagenic in the Salmonella/microsome assay. This observation suggests that there is a causality in the association between increased cancer risk and passive-smoking, as was found by other investigators.

Prediction of overall pulmonary function loss in relation to the 3-D dose distribution for patients with breast cancer and malignant lymphoma.

PURPOSE: To predict the changes in pulmonary function tests (PFTs) 3-4 months after radiotherapy based on the three-dimensional (3-D) dose distribution and taking into account patient- and treatment-related factors. METHODS: For 81 patients with malignant lymphoma and breast cancer, PFTs (VA, VC, FEV1 and TL,COc) were performed prior to and 3-4 months after irradiation and dose-effect relations for early changes in local perfusion, ventilation and air-filled fraction were determined using correlated CT and SPECT data. The 3-D dose distribution of each patient was converted into four different dose-volume parameters, i.e. the mean dose in the lung and three overall response parameters (ORPs, which represent the average local injury over the complete lung). ORPs were determined using the dose-effect relations for early changes in local perfusion, ventilation and air-filled fraction. Correlation coefficients were calculated between these dose-volume parameters and the changes in PFTs. In addition, the impact of the variables chemotherapy (MOPP/ABV and CMF), tamoxifen, smoking, age and gender on the relation between the mean lung dose and the relative changes in PFTs following radiotherapy was studied using multiple regression analysis. RESULTS: The mean lung dose proved to be the easiest parameter to predict the reduction in PFTs 3-4 months following radiotherapy. For all patients the relation between the mean lung dose and the changes in PFTs could be described with one regression line through the origin and a slope of 1% reduction in PFT for each increase of 1 Gy in mean lung dose. Smoking and CMF chemotherapy influenced the reduction in PFTs significantly for VA and TL,COc, respectively. Patients treated with MOPP/ABV prior to radiotherapy had lower pre-radiotherapy PFTs than other patient groups, but did not show further deterioration after radiotherapy (at 3-4 months). CONCLUSIONS: The relative reduction in VA, VC, FEV1 and TL,COc 3-4 months after radiotherapy for breast cancer and malignant lymphoma can be estimated before radiotherapy based on the mean lung dose of each individual patient and taking into account the use of chemotherapy and smoking habits of the patient.

Dose-effect relations for early local pulmonary injury after irradiation for malignant lymphoma and breast cancer.

PURPOSE: To quantify the influence of treatment- and patient-related factors on the severity of early local pulmonary injury and to establish whether regional differences are present for local dose-effect relations for early radiation-induced pulmonary injury. METHODS: Forty-two patients with malignant lymphoma and 40 breast cancer patients were examined prior to and 3 months after radiotherapy. The lymphoma patients were irradiated with mantle fields to an average dose of 38 Gy and the breast cancer patients were irradiated with internal mammary node fields with or without tangential breast fields to an average dose of 50 Gy. Dose-effect relations for local perfusion, ventilation and density changes were determined using correlated single photon emission computed tomography (SPECT) and CT data. A multivariate analysis was performed to study the influence of irradiated volume, chemotherapy (CMF and MOPP/ABV), smoking, age and gender. In addition, dose-effect relations for different regions in the lung were determined. RESULTS: A similar and almost linear increase of early functional changes as a function of radiation dose was observed for perfusion and ventilation, whereas the shape of the dose-effect relation and the magnitude of early structural changes were different for density. For the three end-points studied, regional differences in radiosensitivity could not be demonstrated. For the posterior lung region compared to the anterior lung region, however, a difference was observed, which could be attributed to a gravity-related effect in the measuring procedure. Local structural changes (density) were significantly smaller for smokers (P = 0.002) and young patients (P = 0.007), whereas the CMF chemotherapy regimen given after radiotherapy (P = 0.017) significantly increased the amount of functional changes (perfusion). The magnitude of local pulmonary changes was independent of the irradiated volume, the MOPP/ABV chemotherapy regimen and gender. CONCLUSION: The dose-effect relations for early radiation-induced local pulmonary changes were independent of the irradiated volume, MOPP/ABV, gender and lung region. CMF, smoking and age influenced the magnitude of early pulmonary changes and should be taken into account in dose-escalation protocols.

Evaluation of two dose-volume histogram reduction models for the prediction of radiation pneumonitis.

PURPOSE: To evaluate the similarities between the mean lung dose and two dose-volume histogram (DVH) reduction techniques of 3D dose distributions of the lung. PATIENTS AND METHODS: DVHs of the lungs were calculated from 3D dose distributions of patients treated for malignant lymphoma (44), breast cancer (42) and lung cancer (20). With a DVH reduction technique, a DVH is summarized by the equivalent uniform dose (EUD), a quantity which is directly related to the normal tissue complication probability (NTCP). Two DVH reduction techniques were used. The first was based on an empirical model proposed by Kutcher et al. (Kutcher, G.J., Burman, C., Brewster, M.S., Goitein, M. and Mohan, R. Histogram reduction method for calculating complication probabilities for three-dimensional treatment planning evaluations. Int. J. Radiat. Oncol. Biol. Phys. 21: 137-146, 1991), which needs a volume exponent n. Several values for n were tested. The second technique was based on a radiobiological model, the parallel functional subunit model developed by Niemierko et al. (Niemierko, A. and Goitein, M. Modeling of normal tissue response to radiation: the critical volume model. Int. J. Radiat. Oncol. Biol. Phys. 25: 135-145, 1993) and Jackson et al. (Jackson, A., Kutcher, G.J. and Yorke, E.D. Probability of radiation-induced complications for normal tissues with parallel architecture subject to non-uniform irradiation. Med. Phys. 20: 613-625, 1993), for which a local dose-effect relation needed to be specified. This relation was obtained from an analysis of perfusion and ventilation SPECT data. RESULTS: It can be shown analytically that the two DVH reduction techniques are identical, if the local dose-effect relation obeys a power-law relationship in the clinical dose range. Local dose-effect relations based on perfusion and ventilation SPECT data can indeed be fitted with a power-law relationship in the range 0-80 Gy, from which values of n = 0.8-0.9 were deduced. These correspond to the commonly used value of n = 0.87 for lung tissue and yielded EUDn=0.87 values which were almost identical to the mean lung doses. For other n values, for which no experimental data are present, differences exist between EUD and mean dose values. Six patients with malignant lymphoma (6/44) and none of the breast cancer patients (0/42) developed radiation pneumonitis. These cases occurred only at high values for the mean lung dose. CONCLUSION: The two DVH reduction techniques are identical for lung and are very similar to mean dose calculations. The two techniques are also relatively similar for other model parameter values.

Is there influence of smoking on the mutagenicity of follicular fluid?

The mutagenicity of follicular fluid was examined in 24 patients, 12 smoking and 12 nonsmoking, who were treated in an in vitro fertilization program. The Salmonella microsome assay was used. It was found that the mutagenicity of follicular fluid was not influenced by the number of cigarettes smoked. Urine samples of smoking in vitro fertilization (IVF) patients however showed a dose-dependent elevation of the mutagenicity.

Appearance and reappearance of mutagens in urine from rats after oral administration of direct brown 95, due to coprophagy.

Rats treated orally with direct brown 95, a benzidine-based dye, widely used in dyeing of textiles, plastics, paper and other materials, showed 2 peaks of excretion of mutagenic products in urine, one between 6 h and 18 h after administration and one about 30 h later. Prevention of coprophagy by fitting neck collars resulted in the disappearance of the second peak. Oral administration of carminic acid resulted in a biphasic excretion of this dye in the feces, due to coprophagy. The excretion pattern of mutagens in urine after administration of direct brown 95 corresponds with the excretion pattern in the feces of orally administered carminic acid.

The appearance of mutagens in urine of rats after the administration of benzidine and some other aromatic amines.

The mutagenicity of urine from rats treated with benzidine or 5 other arylamines (0.25 mmol/kg; i.p.) was studied using the Ames-assay. It was found that samples of urine collected for 24 h after the administration of the carcinogens, benzidine, 4-aminobiphenyl and 2-aminonaphthalene, showed significantly mutagenic activity, whereas no mutagenicity was observed in urine after treatment with 3,3'-5,5'-tetramethylbenzidine, 2-aminobiphenyl and 1-aminonaphthalene. Mutagenic activities were dependent on the use of either hepatic S-9 Mix or cytosol as the activating enzyme preparation. The addition of beta-glucuronidase enhanced mutagenicity, except for 2-aminonaphthalene. The appearance of mutagens in urine was studied at varying doses of benzidine and at different time-intervals after the administration. The different excretion patterns found after the activation either with S-9 Mix or with cytosolic enzyme(s) suggest the presence in urine of different types of mutagenic products.

Involvement of non-oxidative enzymes in mutagenic activation of urine from rats, given benzidine and some other aromatic amines.

Urinary metabolites of rats treated with benzidine and some other genotoxic aromatic amines became mutagenic in the Ames assay after activation with liver cytosol from rat, mouse and guinea pig. Most of the mutagenic metabolites appeared in urine as glucuronides. Strong evidence was found that N,O-acyltransferase is responsible for the mutagenic activation by rat liver cytosol. The inhibitory effect of paraoxon and sodium fluoride indicates that the activation by mouse liver cytosol is due to the action of deacetylase. Mutagenic activation by guinea pig liver cytosol seemed to be mediated in part by deacetylase. The metabolite activated by these enzymes most likely is a glucuronidated, N-acetylated, N-hydroxylated product.

Metabolism of benzidine-based dyes and the appearance of mutagenic metabolites in urine of rats after oral or intraperitoneal administration.

The azo reduction and acetylation in vitro and the mutagenic activation in vivo of three azo dyes were studied. In the presence of rat-liver 9000 g supernatant benzidine was released from direct black 38 and direct brown 95, whereas hardly any benzidine was produced during incubation of direct blue 6. Incubation of benzidine with isolated rat hepatocytes resulted in the appearance of diacetylbenzidine. No diacetylbenzidine was formed during incubation of benzidine with rat-liver 9000 g supernatant, unless the cofactor for the acetylation reaction, acetyl coenzyme A, was added to the incubation medium. Isolated rat hepatocytes were capable to produce diacetylbenzidine from direct black 38, direct blue 6 or direct brown 95 without supplementation with acetyl coenzyme A. Administration of benzidine, direct black 38 or direct brown 95 to rats resulted in the appearance of mutagenicity in urine. For direct black 38 significantly higher mutagenicity values were found in urine after oral administration than after intraperitoneal treatment. Such differences were not observed for benzidine and direct brown 95. The results demonstrate that rat liver has a considerable capacity to reduce azo compounds. Nevertheless, for some compounds, like direct black 38, extrahepatic enzymes, most likely present in the intestinal flora, may also play a substantial role in the azo cleavage.

Internal exposure of rats to benzidine derived from orally administered benzidine-based dyes after intestinal azo reduction.

The role of the rat intestinal flora in the azo reduction of some benzidine-based dyes was studied in vitro and in vivo. The formation of benzidine was measured after anaerobic incubation of direct black 38, direct blue 6 and direct brown 95 in the presence of caecal bacteria in vitro. Benzidine was absorbed from the intestinal tract much better than the parent compounds. Oral administration of direct black 38 or direct brown 95 to Wistar rats results in the urinary excretion of mutagens. After oral administration of these dyes to germ-free Wistar rats no mutagenicity was observed in urine. The present results show that after oral administration, reduction by the intestinal flora should be considered as the first essential step in the biotoxification of benzidine-based dyes.

Excretion of benzo[a]pyrene and metabolites in urine and feces of rats: influence of route of administration, sex and long-term ethanol treatment.

The urinary and fecal excretion of benzo[a]pyrene (B[a]P) and its main metabolites were studied after oral and intraperitoneal administration of B[a]P to male and female ethanol-treated and non-ethanol-treated rats. After oral administration of B[a]P more mutagenic compounds as well as B[a]P metabolites were found in feces than after intraperitoneal administration. The excretion of B[a]P metabolites in urine and feces after oral administration were maximal at days 1 and 2 whereas after intraperitoneal administration excretion was maximal at days 2 and 3. In males, the amounts of excreted phenolic metabolites in urine and feces were generally higher than in females. The amounts of mutagenic products in urine and feces of males were also higher than in females after intraperitoneal and oral administration of B[a]P. In urine of female rats that received B[a]P intraperitoneally, a decreased excretion of phenolic metabolites was found after ethanol treatment. In feces of both male and female rats, a decreased excretion of 3-OH-B[a]P was found after ethanol treatment. In this study, the influence of sex and administration route on the excretion of B[a]P metabolites was more pronounced than the effect of ethanol treatment.

In vitro/in vivo effects of Mesna on the genotoxicity and toxicity of cyclophosphamide--a study aimed at clarifying the mechanism of Mesna's anticarcinogenic activity.

Cyclophosphamide is an effective antitumor agent with considerable side effects such as urotoxicity and carcinogenicity. These negative attributes may be caused by toxic and genotoxic metabolites, respectively. Mesna (sodium 2-mercaptoethane sulfonate) decreases the urotoxicity by scavenging the toxic metabolite acrolein. The present study was aimed at elucidating whether a similar scavenging of genotoxic alkylating intermediates could be found, which might cause the reduction in carcinogenicity. In vitro studies on the genotoxic and toxic properties of cyclophosphamide and its major metabolites to bacteria were therefore performed in the presence of Mesna. Mesna did not reduce the mutagenicity of any of the tested metabolites. Mesna clearly inhibited the toxic properties of acrolein. After in vivo application of Mesna and cyclophosphamide to rats, however, a lower yield of mutagens in the excreted urine was observed than after application of cyclophosphamide only.

Mutagenicity of urine from rats after 1-nitropyrene and 2-nitrofluorene administration using new sensitive Salmonella typhimurium strains YG1012 and YG1024.

1-Nitropyrene (1-NP) and 2-nitrofluorene (2-NF), two of the most abundant nitro-substituted polycyclic aromatic hydrocarbons (nitro-PAH) present in combustion products such as diesel engine exhaust, were administered intraperitoneally to rats at a dose of 5 mg per animal. Urine samples, 1-NP and 2-NF were tested in the Ames assay using the newly developed Salmonella typhimurium strains YG1012 and YG1024 (overproducing O-acetyltransferase) and their parent strains TA1538 and TA98. In urine, collected over 3 periods of 24 h after administration, most of the mutagens appeared during the first 24 h. The mutagenicity was found to be a factor 2-30 higher in the YG strains when compared to the TA strains. Addition of S9 mix and rat liver cytosol both with and without beta-glucuronidase increased the mutagenicity of urine samples from 1-NP-treated rats. Addition of beta-glucuronidase revealed that a considerable part of the mutagenic metabolites of 1-NP and 2-NF were excreted as glucuronide conjugates. The increase in mutagenicity of urine samples from 2-NF-treated rats after the addition of rat liver cytosol referred to N,O-acyl transfer as a step in activating 2-NF to strong mutagens. The high sensitivity of the YG tester strains indicated that these strains might be used to explore environments where people are exposed to nitro-PAH, such as work places with diesel emission sources.