The utility of PBPK in the safety assessment of chloroform and carbon tetrachloride.

Occupational exposure limits (OELs) for individual substances are established on the basis of the available toxicological information at the time of their promulgation, expert interpretation of these data in light of industrial use, and the framework in which they sit. In the United Kingdom, the establishment of specific OELs includes the application of uncertainty factors to a defined starting point, usually the NOAEL from a suitable animal study. The magnitude of the uncertainty factors is generally determined through expert judgment including a knowledge of workplace conditions and management of exposure. PBPK modeling may help in this process by informing on issues relating to extrapolation between and within species. This study was therefore designed to consider how PBPK modeling could contribute to the establishment of OELs. PBPK models were developed for chloroform (mouse and human) and carbon tetrachloride (rat and human). These substances were chosen for examination because of the extent of their toxicological databases and availability of existing PBPK models. The models were exercised to predict the rate (chloroform) or extent (carbon tetrachloride) of metabolism of these substances, in both rodents and humans. Monte Carlo analysis was used to investigate the influence of variability within the human and animal model populations. The ratio of the rates/extent of metabolism predicted for humans compared to animals was compared to the uncertainty factors involved in setting the OES. Predictions obtained from the PBPK models indicated that average rat and mouse metabolism of carbon tetrachloride and chloroform, respectively, are much greater than that of the average human. Application of Monte Carlo analysis indicated that even those people who have the fastest rates or most extensive amounts of metabolism in the population are unlikely to generate the levels of metabolite of these substances necessary to produce overt toxicity in rodents. This study highlights the value that the use of PBPK modeling may add to help inform and improve toxicological aspects of a regulatory process.

Dermal uptake of solvents from the vapour phase: an experimental study in humans.

The control of exposure to hazardous substances in the workplace has traditionally focused on uptake via the inhalation route. Control of skin uptake has generally been considered for solids and liquids but the potential for uptake from vapours and gases has received relatively little attention. The current work was undertaken to establish a methodology to study the dermal uptake from vapours and to provide new and comparative information on a range of substance vapours. Groups of human volunteers were exposed to a small range of substances either 'whole body' or via the skin only. Substances (xylene, toluene, tetrahydrofuran [THF], methyl ethyl ketone [MEK] and 1-methoxypropan-2-ol [M2P]) were selected on the basis of their predicted dermal uptake from the vapour phase; their industrial use and potential for occupational exposure; the existence of a health-based occupational exposure limit; the availability of an analytical technique(s) for the substance and/or metabolite(s); and as representatives of chemical classes. Exposures were for four hours generally at the level of the UK Occupational Exposure Standard. Uptake was assessed by monitoring of parent or metabolite in blood, single breath or urine following exposure. Uptake of xylene, toluene and THF vapours via the skin under the conditions of this study was estimated to contribute around 1-2% of the body burden received following whole body (including inhalation) exposure. MEK showed more uptake via the skin, contributing around 3-3.5% of the body burden. Most dermal uptake was seen for the glycol ether M2P for which estimates of between 5-10% of whole body exposure body burden were obtained. The results of this and other studies indicate that uptake of vapours across the skin can occur but that for some substances (e.g., xylene, toluene, THF) this is likely to contribute little to the body burden. For other substances, such as the glycol ethers, skin uptake from vapours may be an important contributor to total uptake, particularly in situations where respiratory protective equipment is used to control inhalation exposure.

Bunyaviridae. Serological relationships.

The family Bunyaviridae comprises 5 genera of lipid-enveloped viruses with trisegmented RNA genomes. One of the genera, Bunyavirus, comprises over a quarter of the known arboviruses. The members of the Hantavirus genus are apparently the exception in that they are not arthropod-borne. The genera are sorted largely on serological grounds. The Nairoviruses, in addition to the presence of the physical marker of a higher molecular weight nucleoprotein, include Congo-Crimean haemorrhagic fever, an apparently isotypic virus of wide distribution.

Protection from radiation-induced damage to spermatogenesis in the androgen pretreated rat.

Protection of spermatogenesis from radiation-induced damage has been investigated in the adult Wistar rat. Silastic tubing containing either cholesterol or testosterone was implanted subcutaneously 7 weeks before 4 equal daily fractions of either 1, 1.5, 2, and 2.5 Gy of 230 kVp X-rays locally to the testes. Implants were removed on the day following the last fraction and 8 weeks after irradiation 88.6%, 83.8%, 63.6% and 28.9% tubule cross-sections respectively were found regenerating in rats pretreated with testosterone. In contrast, 68.45%, 58.6%, 38.2% and 17.3% tubule cross-sections regenerating were obtained in rats pretreated with cholesterol. Changes in testis weight however were found to show the reverse trend (i.e. a greater weight loss was observed following androgen pretreatment). These results show that protection of spermatogenesis from fractionated irradiation may be achieved in rat testis by androgen pretreatment.

Growth and chemotherapy of a human germ-cell tumour line (GCT 27).

The human germ-cell tumour cell line GCT 27 growing as subcutaneous xenograft tumours in male nude mice was used in the 4th and 5th passage to study chemotherapeutic drug responses. Recipient mice received 5 Gy whole body irradiation immediately before tumour transplantation. The median take rate was 62% (range 39-73%) and the median volume doubling time 14 days (range 7-28 days). For bleomycin, cisplatin and carboplatin a clear dose response for growth delay was observed. Bleomycin caused substantial weight loss at doses above 75 mg/kg whereas good response to cisplatin was obtained without serious toxic effects. Vinblastine and etoposide exerted no effect when given in non-toxic doses. The response to etoposide was not improved either by fractionated treatment or by combination with verapamil. However, the combination of 20 mg/kg etoposide and 2 mg/kg cisplatin, which when given alone were ineffective, led to a growth delay that was equal to that observed following the administration of higher cisplatin doses. This effect may be explained by the fact that etoposide, as an inhibitor of DNA-topoisomerase II, may interfere with the repair of DNA interstrand cross-links caused by cisplatin.

Comparative effects of X irradiation on the testes of adult Sprague-Dawley and Wistar rats.

The response of the testes of two strains of adult rats (Sprague-Dawley and Wistar) to graded single doses and split doses of 230 kVp X rays has been investigated. A marked difference was noted between the strains in the response of the clonogenic spermatogonia to irradiation, as measured histologically by the repopulation index. Single-dose response curves derived for these cells in the Sprague-Dawley strain had a much larger shoulder (up to about 4-5 Gy) than for the Wistar (less than 2 Gy). Split-dose studies revealed that this difference may partly be explained by a greater repair capacity in the cells of the Sprague-Dawley strain. Changes in serum FSH concentrations mirrored the changes in clonogenic spermatogonial survival following split doses of radiation.

The response of murine stem spermatogonia to radiation combined with 3-aminobenzamide.

The influence of 3-aminobenzamide (3-AB) on the radiation response of the stem spermatogonia of the CBA mouse has been investigated. Doses of 3-AB from 66 to 450 mg/kg, administered 1 h before irradiation, significantly enhanced stem-cell killing. Enhancement was observed when 3-AB (450 mg/kg) was given up to 5 h before, but not if administered after, irradiation. When radiation was delivered at a lower dose rate (5 cGy/min compared to 180 cGy/min) significant dose sparing was achieved for radiation alone. Pretreatment with 3-AB resulted in slightly less enhancement at the low dose rate than at the high. Split-dose studies (9 Gy total dose) with radiation alone resulted in a recovery ratio of 1.4-1.5. Administration of 3-AB before the first dose resulted in a similar recovery ratio, but if given immediately after the first dose the ratio was smaller. Pretreatment of mice with the radiosensitizer RSU-1069 indicated that at least some of the stem cells were radiobiologically hypoxic. We suggest therefore that the enhancement of spermatogonial stem-cell killing by 3-AB is not entirely due to inhibition of repair processes but may also involve modification of the oxygen status of the testis.

Effect of dose-rate on the survival of murine spermatogonia following 60Co gamma-irradiation.

The survival of murine clonogenic stem and differentiated spermatogonia was assessed by counts of repopulating tubules in histological sections at 35 days and counts of testicular sperm-heads at 29 days respectively, following irradiation delivered at five dose-rates (1.1, 2.5, 5, 11 and 112 Cgy X min-1). For stem spermatogonia, a significant increase in cell survival accompanied the reduction in dose-rate, and this was characterized by an increase of the Do for the derived survival curves from 2.86 +/- 0.29 Gy at 112 cGy X min-1 to 4.46 +/- 0.45 Gy at 1.1 cGy X min-1. The data were analysed using two mathematical models for repair at varying dose-rates, the LPL model of Curtis and the incomplete repair model of Thames. Using these models the half-time for repair was estimated to lie between 15 and 25 min. No dose-rate effects were observed for the differentiated spermatogonia. The sparing of stem spermatogonia at reduced dose-rate, may have implications for the future fertility of men treated with low dose-rate total body irradiation (TBI).

Time dependence for the protective effect of androgen from procarbazine-induced damage to rat spermatogenesis.

The protective effects of androgen pretreatment on the procarbazine-induced killing of spermatogonial stem cells in Wistar rats have been investigated. Using testosterone-filled Silastic implants (200 mm2) the degree of protection from four weekly doses of procarbazine (100 mg/kg) was found to be dependent upon the androgen pretreatment time interval as assessed by quantitative histology. No protective effect was seen until rats had received 4 wk of pretreatment with androgen, whereafter protection increased to a maximum (about 20 to 30% of tubule cross-sections exhibiting recovery) after 8 to 12 wk of pretreatment. In contrast, the same level of maximal protection could be obtained by 6 wk of pretreatment using testosterone enanthate, suggesting that differences in protection may be achieved using different modes of androgen administration.

Attempted protection of spermatogenesis from single doses of gamma-irradiation in the androgen pretreated rat.

Spermatogenic stem-cell survival after gamma-irradiation has been investigated in the adult Wistar rat. Single doses of 4.5 and 9 Gy gamma-rays were administered to the testes of rats who received arachis oil (0.1 ml/100 g body weight) or testosterone enanthate (240 micrograms/100 g body weight) subcutaneously three times weekly for 6 weeks prior to radiation and during the week in which the radiations were given. A mean percentage of regenerating seminiferous tubule cross-sections of 32.45% and 7.26% was found in the testes of androgen-pretreated rats at 8 weeks after 4.5 and 9 Gy, respectively. Similar values (33.4% and 6.2%) were obtained in arachis oil-pretreated controls. We therefore conclude that protection of rat spermatogenesis from single doses of gamma-rays cannot be achieved by androgen pretreatment.

Protection of spermatogenesis during cancer chemotherapy.

The ability of androgen pretreatment in adult Wistar rats to protect the spermatogonial stem cells from one or two doses of the cytotoxic procarbazine (PCB) was investigated. Survival of stem cells was assessed by a tubule regeneration assay. Ten weeks' pretreatment of the rats with 200 mm2 silastic implants containing testosterone resulted in significant protection of the spermatogonial stem cells from PCB-induced damage. Androgen-pretreated rats given a single dose of 200 mg/kg PCB demonstrated 86% tubule regeneration as opposed to 41% in rats pretreated with cholesterol. Similar values were obtained when the PCB dose was given as two weekly fractions of 100 mg/kg. The protective effect of androgen is present from the time of the initial challenge with the cytotoxic drug.

Influence of timing of cytotoxic drug treatment on the response of murine clonogenic spermatogonia to X-irradiation.

The enhancement of radiation-induced stem spermatogonial cell killing by three cytotoxic compounds has been investigated using an in vivo clonogenic assay. Drug doses (carmustine (BCNU), 15 mg/kg; cyclophosphamide (CY), 150 mg/kg; procarbazine (PCB), 200 mg/kg) were chosen to kill a high proportion of differentiated spermatogonia but few stem-cells, as assessed by sperm-head counts (SHC) at 29 and 56 days respectively. Drug doses were administered up to 14 days prior to or after local irradiation with 9 Gy of 230 kVp X-rays. BCNU produced a moderate enhancement of damage that was relatively independent of time. Both CY and PCB produced a considerable enhancement of damage especially when administered 1 to 3 days before irradiation. Enhancement in the case of PCB consisted largely of a vertical displacement of the radiation dose-response curves, but with additional evidence for possible changes in slope.

Survival of murine clonogenic stem spermatogonia following combined radiation and cytotoxic drug treatments.

The effects of combined radiation and cytotoxic drug treatments on the survival of murine clonogenic stem spermatogonia have been investigated using an in vivo clonogenic assay. Drug doses (BCNU, 15 mg/kg; cyclophosphamide (CY) 150 mg/kg, and procarbazine (PCB) 200 mg/kg) were chosen to kill a high proportion of differentiated spermatogonia, but no stem-cells, and were administered as single doses up to 14 days prior to or after 9 Gy of X rays. BCNU produced a moderate enhancement of damage (DEF, 1.0-1.3) that was relatively time-independent. Both CY and PCB demonstrated considerable enhancement of damage (DEF, 1.6 +/- 0.03, respectively) when administered 1 day prior to radiation, and also 3 days prior to radiation for PCB (DEF = 1.4 +/- 0.05). The enhancement of damage may have been caused by either a change in the shoulder width of the cell-survival curve, or additive cell-killing as indicated by a dose-response study.

Protection from procarbazine-induced damage of spermatogenesis in the rat by androgen.

Protection of spermatogenesis from cytotoxic drug-induced damage has been investigated in the rat. Complete germinal aplasia was observed at 8 and 11 weeks after four doses of procarbazine hydrochloride administered weekly (150 mg/kg, first dose; 100 mg/kg, 3 subsequent doses). Pretreatment of rats for 6 weeks plus continued treatment during procarbazine administration with testosterone enanthate, 240 micrograms/100 g body weight, resulted in a marked protection of spermatogenesis. A mean of 22% of seminiferous tubule cross-sections at both 8 and 11 weeks exhibited spermatogenesis, and developing spermatids were observed in 60% of these repopulating tubules at the later time. These results provide evidence that protection of spermatogenesis during chemotherapy may be achieved by androgen treatment.

Seminiferous epithelial function in the pubertal rat following local testicular irradiation.

The dose- and time-related responses of the irradiated seminiferous epithelium in the pubertal rat have been investigated. The threshold dose for Sertoli cell dysfunction, as assessed by serum androgen binding protein (ABP) concentrations, was estimated to be 5 Gy. A significant reduction (to less than 50% of control levels) in serum ABP was observed at 8 weeks post-irradiation, with further reductions at later times (24 and 36 weeks). Serum follicle-stimulating hormone (FSH) was elevated to between 130 and 175% of control at only 2 weeks post-irradiation, but recovered with time. Normal FSH levels seemed to be related to recovery of spermatogenesis, as assessed by counts of regenerating tubule cross-sections. The results indicate that the clonogenic spermatogonia and Sertoli cells of the pubertal rat testis are less sensitive to radiation than those of the adult.

Testicular function in adult rats treated with the alkylating agent chlorambucil.

The effects of multiple treatments with the alkylating agent chlorambucil on testicular function in the adult Wistar rat were evaluated. Weekly treatment with doses of 2.5, 5, or 10 mg/kg produced no effect either on spermatogenesis or Leydig cell function. In contrast, doses of 8 or 10 mg/kg administered twice weekly induced damage to both spermatogenesis and probably the Leydig cells. A dose-dependent decrease in spermatogonial stem-cell survival was observed with these two regimens, as assessed by counts of repopulating tubule cross sections. Although serum testosterone remained unchanged, possible Leydig cell damage was indicated by an approximate twofold increase in serum LH following treatment with either 8 or 10 mg/kg of chlorambucil twice weekly. The present results demonstrate that multiple treatments with cytotoxic drugs can be used to model the testicular damage observed in man.

Leydig cell function in the pubertal rat following local testicular irradiation.

Dose- and time-response relationships were measured after irradiation of the pubertal rat testis with between 1 and 20 Gy of 300 kVp X-rays. The threshold dose for Leydig cell dysfunction was about 5 Gy. Dysfunction after higher doses was observed by 2 weeks post-irradiation as a dose-dependent decrease in serum testosterone (T) concentrations, and the levels were undetectable after 15 or 20 Gy. Despite the recovery of serum T by 24 and 36 weeks, dysfunction of the Leydig cell population was still observed as an increase in luteinizing hormone (LH) secretion (1.5 to 2-fold increase after 15 or 20 Gy at 24 weeks; 2 to 3-fold increase after 10 or 20 Gy at 36 weeks). The endocrine changes were probably due to the observed loss of Leydig cells following irradiation. These results indicate that the Leydig cells of pubertal rats are more radioresponsive than those of the adult.

Dose and time relationships in the endocrine response of the irradiated adult rat testis.

The dose- and time-dependent responses for the interstitial and tubular compartments in irradiated adult rat testes are described. Leydig cell dysfunction, as indicated by increased serum LH (to a maximum of 385% of control after 5 Gy) and decreased serum T (to a minimum of 30% of control after 10 Gy), was observed at 8 weeks postirradiation. Subsequent recovery of Leydig cell function was then observed, so that after 9 months serum T was normal but LH was still marginally elevated. The dysfunction, with a threshold of about 4 to 5 Gy, was associated with a loss of Leydig cells from the testis. Spermatogenic damage was observed; after doses of 3 Gy and above a marked dose-response was recorded as assessed by counts of tubule cross sections exhibiting spermatogenesis. Reduced serum levels of androgen binding protein indicated Sertoli cell dysfunction at 8 weeks after 3 Gy and above, with values of less than one half of those seen in the controls. Serum FSH also was elevated to between 150% and 200% of control, and after 9 months closely reflected androgen binding protein changes. Unlike the Leydig cell, no recovery with time was observed for this aspect of Sertoli cell function.