Identification of an alginate-based formulation of paraquat to reduce the exposure of the herbicide following oral ingestion.

The herbicide paraquat has been widely used throughout the world for almost 50 years and is important in sustainable agriculture. When used correctly the chemical poses no known risk to human health. However, it is acutely toxic, and can be fatal, if the concentrated product is ingested orally. Despite many years of research there is no successful treatment for paraquat intoxication. In recent years we have turned our attention to understanding how we can make the product safer, if it is accidentally or intentionally consumed. We present in this paper a novel approach aimed at safening the paraquat product, Gramoxone. Following our previous research on the site and mechanism of paraquat absorption from the gastrointestinal tract we have identified a new formulation of paraquat, Gramoxone INTEON that reduces the absorption of paraquat into the blood. This new formulation contains the polysaccharide, alginate, a natural product extracted from sea-weed. We have designed a preparation of paraquat and alginate with surfactants that is herbicidally active but has the unique property that it gels on contact with gastric acid in the stomach. The resulting mixture slows the dispersion and delivery of the toxic chemical to its site of absorption in the small intestine. Alginates also protect the mucosa against the damaging influence of topical gastric irritants, like paraquat. Our studies have shown that increasing the loading of alginate between 7 and 17 g/L causes a dose-related reduction in paraquat absorption in vitro in isolated rat ileum. This is also observed in vivo, as measured by paraquat plasma kinetics in the rabbit where the Area Under Curve (AUC 0-24h) was reduced from 33.8+/-3 for Gramoxone to 12.5+/-6 (microg/mL)h for a formulation containing 17 g/L alginate. Such a reduction in systemic exposure to paraquat is expected to reduce the acute oral toxicity of the formulation. This should be particularly effective in a vomiting species such as man since we have shown in this investigation that alginates not only reduce the peak plasma paraquat values but also delay the time to peak levels. This provides the opportunity for a more effective emetic response since the highly viscous gelled material should remain in the stomach for longer than the liquid Gramoxone. Further research is required to understand and optimise the safening and herbicidal characteristics of these alginate acid-triggered gel formulations of paraquat. However, we anticipate that this alginate technology in Gramoxone INTEON could have significant benefit in reducing human mortalities associated with the herbicide.

Acetochlor-induced rat nasal tumors: further studies on the mode of action and relevance to humans.

The herbicide acetochlor, and its analogue alachlor, have similar toxicological properties, the most significant being the induction of nasal adenomas in rats in 2-year feeding studies. Previous investigations have proposed a mode of action involving metabolism to a quinone-imine, the formation of protein adducts, cell death, and compensatory hyperplasia leading to the observed adenomas. Comparisons between rats and humans of the metabolic cascade leading to the quinone-imine indicate that these chemicals do not pose a threat to humans. Further investigations with acetochlor, presented here, have revealed an additional activation pathway in which a sulfoxide metabolite of acetochlor plays a key role. The sulfoxide was found to be the major plasma metabolite in rats dosed with acetochlor. Whole-body autoradiography studies established that this metabolite selectively accumulates and persists in the olfactory epithelium of rats. Radiolabeling of the sulfoxide molecule in the phenyl ring and in the sulfoxide side-chain demonstrated that the metabolite accumulating in nasal tissues retains the sulfoxide side-chain. The formation of a quinone-imine from the sulfoxide was facilitated by hydroxylation of the phenyl ring by a cytochrome P450 isoenzyme which was specific to the nasal epithelium in the rat. This metabolic conversion could not be detected in 33 fresh human nasal tissue samples, supporting the earlier view that the acetochlor-induced rat nasal tumors do not represent a hazard for humans.

Evaluation of the mutagenicity of acetochlor to male rat germ cells.

Male rat dominant lethal (DL) assays conducted on the herbicide acetochlor are described. Single dose studies conducted at the maximum tolerated dose (MTD, < or = 1000 mg/kg) produced no effects on any of the DL assay parameters at any of the ten weekly sampling periods. It is concluded that acetochlor is non-mutagenic to rat germ cells. Due to initial limited knowledge of the MTD of acetochlor it was also evaluated in the DL assay at a dose level of 2000 mg/kg. At this high dose level severe bodyweight loss and some deaths occurred among the treated animals. In addition, reduced implantations and reduced pregnancy rates were observed at the third sampling period (18-25 days post dosing) in the absence of an increase in early post-implantation deaths. These results indicated that the use of supra-MTD doses of acetochlor had reduced the fertility of the treated males leading to the production of a pseudo-DL assay response, as alerted to and defined by Ehling. Although several such pseudo-DL assay responses have been described, none have been explained mechanistically. It was therefore decided to pursue the effects seen in the DL assay when using supra-MTD doses of acetochlor. Ova analysis of female rats mated with male rats exposed to 2000 mg/kg acetochlor revealed unfertilized ova at the critical third sampling time. Normal fertilization of ova was observed at the first and fifth sampling period and, for a dose of 200 mg/kg acetochlor, at the third sampling period. The magnitude and temporal nature of these effects confirmed the induction of a pseudo-DL assay response, and studies were then undertaken to probe its genesis. Rats treated with 2000 mg/kg acetochlor had normal testicular and epididymal pathology and normal sperm numbers and sperm motility at the critical third sampling period. Despite a small reduction in testicular and epididymal glutathione levels 12 h after exposure to 2000 mg/kg acetochlor, testicular LDH and LDH-X enzyme levels were unaffected. Further, no reduction in the level of free sulphydryl groups (-SH) were observed in epididymal caput sperm heads isolated 0.5, 7 or 14 days after treatment of male rats with 2000 mg/kg acetochlor. The only sperm parameter affected by treatment with 2000 mg/kg acetochlor was an increase in epididymal cauda sperm with head abnormalities. The non-specific nature of this effect was considered inadequate to explain fully the high dose fertility effects seen in the DL assays, which therefore remain unexplained. The present data establish that acetochlor is non-mutagenic to rat germ cells. They also confirm the importance of segregating mutagenic and fertility effects in the DL assay, and emphasize the need for appropriate dose-setting studies prior to the conduct of rodent genetic toxicity assays.

Methyl methanesulphonate (MMS) as a resource conserving and reliable positive control agent for male rat and male mouse dominant lethal assays.

A single i.p. injection of methyl methanesulphonate (MMS; 40 mg/kg) to male rats, followed by their sequential mating over 4-28 days post-dosing, was shown to induce dominant lethal (DL) effects in two strains of rats and in four separate experiments. Activity was evident between 4 days post-dosing (sampling treated spermatozoa) and 28 days post-dosing (sampling treated early spermatids). The highest incidences of resorptions were seen between days 15 and 21 post-dosing, being approximately 1 week later than the peak of activity in the mouse. The DL effects seen were strong and could be detected as early as 4-10 days post-dosing, at which time only small reductions in pregnancy rates and total implantation numbers were seen. Similar, but weaker, mutagenic effects have been reported by Ehling et al. for MMS in mouse DL assays. These data indicate, therefore, that MMS provides a convenient positive control agent for both male rat and male mouse dominant lethal assays, requiring only approximately five treated males, each mated to two females at around 14 days post-dosing.

Evaluation of the potential carcinogenicity and genetic toxicity to humans of the herbicide acetochlor.

Comprehensive toxicological studies of the herbicide acetochlor are presented and discussed. Although it gave a negative profile of responses in the many toxicity tests conducted there were some findings that prompted further investigation. First, although non-mutagenic in the Salmonella assay, acetochlor was clastogenic to mammalian cells treated in vitro. This clastogenic potential was not expressed in vivo in four rodent cytogenetic assays (bone marrow and germ cells). Second, although acetochlor gave a negative response in rat liver UDS assays when tested at the acute MTD, gavage administration of a single, supra-MTD dose (2000 mg/kg) gave a weak positive assay response. This dose-level (2000 mg/kg) was necrotic to the liver, depressed hepatic glutathione levels by up to approximately 80%, altered the metabolism of acetochlor, and was associated with up to 33% lethality. In contrast, reference liver genotoxins such as DMN, DMH and 2AAF were shown to elicit UDS in the absence of such effects, and at approximately 400 x lower dose-levels. Finally, microscopic nasal polypoid adenomas were induced in the rat when acetochlor was administered for two years at the maximum tolerated dose (MTD). The tumours were not life-threatening, they did not metastasize, and no DNA damage was induced in the nasal cells of rats maintained on a diet containing the MTD of acetochlor for either 1 or 18 weeks (comet assay). In order to probe the mechanism of action of these high dose toxicities a series of chemical and genetic toxicity studies was conducted on acetochlor and a range of structural analogues. These revealed the chloroacetyl substructure to be the clastogenic species in vitro. Although relatively inert, this substituent is preferentially reactive to sulphydryl groupings, most evidently, to glutathione (GSH). Similar chemical reactivity and clastogenicity in vitro was observed for two related chemicals bearing a chloroacetyl group, both of which have been defined as non-carcinogens in studies reported by the US.NTP. These collective observations indicate that the source of the clastogenicity of acetochlor in vitro is also the source of its rapid detoxification in the rat in vivo, via reaction with GSH. Metabolic studies of acetochlor are described which reveal the formation of a series of GSH-associated biliary metabolites in the rat that were not produced in the mouse. The metabolism of acetochlor in the rat changes with increasing dose-levels, probably because of depletion of hepatic GSH. It is most likely that a rat-specific metabolite is responsible for the rat nasal tumours observed uniquely at elevated dose-levels. The absence of genetic toxicity to the nasal epithelium of rats exposed acutely or subchronically to acetochlor favours a non-genotoxic mechanism for the induction of these adenomas. The observation of a time- and dose-related increase in S-phase cells in the nasal epithelium is consistent with this conclusion. Despite some confusion caused by the early use of perilethal gavage administrations of acetochlor to rodents, and supra-MTD dietary concentrations in some of the chronic studies, the available MTD data are consistent with acetochlor not posing a genetic or carcinogenic hazard to humans.

Fate of unfertilized ova in male rodent dominant lethal assays: extension of the studies by Kratochvilova.

Kratochvilova has described a technique whereby ova can be recovered from mated mice and their stage of division determined. This is of value to determine if reduced total implantations in a male dominant lethal (DL) germ cell mutation assay are due to pre-implantation loss of embryos, a presumed mutagenic event, or to chemically induced male infertility. Kratochvilova was not specific about the fate of unfertilized ova, but it was implied that they undergo a process of fragmentation that might be confused with the regular cleavage of fertilized ova. It became important for us to draw a firm distinction between ova fragmentation and regular ova cleavage in the rat. We therefore repeated the ova analyses of female mice mated with males exposed to iso-propyl methanesulphonate (iPMS), as described by Kratochvilova. Following that calibration study the technique was extended to the rat via ova cleavage analysis in mated female rats, coupled to a study of the normal decay of ova in virgin rats. Unfertilized ova are shown to undergo irregular fragmentation that can be clearly distinguished from normal cell division. It is concluded that the individual or combined incidences of single celled ova and fragmented ova (dependent on the cleavage stage of the concurrent control embryos) can provide a measure of male infertility as it relates to reduced implantations in DL assays. This ability to regard two morphological classifications of unfertilized ova as providing evidence for male infertility will simplify the conduct of ova analyses in both the mouse and the rat.

The rodent dominant lethal assay: a proposed format for data presentation that alerts to pseudo-dominant lethal effects.

The rodent dominant lethal (DL) germ cell mutagenicity assay is the primary test for possible human germ cell mutagens. As such, it occupies a critical regulatory position. DL assay data are often difficult to assess because of the quantity of data involved, and because several related assay parameters require to be considered simultaneously. To reduce this difficulty a schematic method of data presentation is proposed and illustrated. This method enables the most pertinent assay data and parameters to be viewed and considered simultaneously. Using this format of data presentation, existing DL studies on cyclophosphamide, methylnitrosourea, diethylhexylphthalate, divinyl sulphone, methyl methanesulphonate, 6-mercaptopurine and ethylenethiourea are re-analysed.

Growth and longevity of rats fed an agar-bound diet.

A conventional diet fed to rats in an agar-gel base reduced mature bodyweight by about 25% and increased longevity when compared to the same diet in pelleted form. Dry matter intake was not affected, but food utilization was considerably poorer. The use of a diet in agar gel may, therefore, provide an improved toxicological assay, especially when used in combination with flushing racks housing 2 animals per cage, where exposure of the animal technician to dust was reduced.

Irradiated laboratory animal diets: dominant lethal studies in the mouse.

In 4 separate dominant lethal experiments groups of mice of either Charles River CD1 or Alderley Park strains were fed laboratory diets (Oakes, 41B, PRD, BP nutrition rat and mouse maintenance diet No. 1). The diets were either untreated (negative control diets) or irradiated at 1, 2.5 and 5 megarad and were freshly irradiated, or stored. The animals were fed their test diets for a period of 3 weeks prior to mating. Groups of mice given a single intraperitoneal injection of 200 mg cyclophosphamide per kg body weight served as the positive controls. Freshly irradiated PRD diet fed to male mice of both strains caused an increase in early deaths in females mated to the males in week 7 and to a lesser extent in week 4. The increase due to irradiation was small by comparison with that produced by the positive control compound. The responses for the other irradiated diets showed no significant increases in early deaths although some values for Oakes diet were high. The effect of storage was examined with PRD and BPN diet on one occasion and produced conflicting results. Thus there was some evidence that irradiated PRD diet has weak mutagenic activity in the meiotic and/or pre-meiotic phase of the spermatogenic cycle which appeared to be lessened on storage; the inclusion of such a diet in toxicological studies would therefore need to be carefully considered.

The effect of diet on some parameters measured in toxicological studies in the rat.

The bodyweight, food consumption and various biochemical and haematological parameters were measured in, and breeding and histological studies made of, groups of rats fed 6 different diets. All diets acceptably supported reproduction. 1 diet restricted bodyweight gain by 30% and increased plasma alanine transaminase activity. Nephrocalcinosis was seen in females fed diets with a calcium:phosphorous ratio of < 1. Levels of dietary protein were positively correlated with the incidence of renal pelvic dilatation in offspring at 7 weeks of age.

Effect of glycerol on local and systemic carcinogenicity of topically applied tobacco condensate.

When glycerol was added to tobacco smoke condensate in acetone solvent, the topical carcinogenicity and the ability to produce epithelial hyperplasia in mice was reduced. Two doses of condensate were applied, combined with 2 concentrations of added glycerol. Age-standardized results show that glycerol reduced the incidence of tumours and malignant tumours and of hyperplasia in animals not developing skin tumours. The relative incidences of malignant tumours, benign tumours, hyperplasia and unaffected skin suggest that there is a sequential relationship (i.e. normal skin to hyperplasia to benign neoplasia to malignant neoplasia) which is impeded by glycerol. There was no systemic effect attributable to the condensate.

Studies on the local and systemic carcinogenicity of topically applied smoke condensate from a substitute smoking material.

The topical carcinogenicity to mouse skin of smoke condensates obtained from a tobacco substitute (NSM), alone or in combination with tobacco, has been compared with condensate from tobacco and with acetone, the solvent used. Sixteen different types of cigarette were used to make the condensates, and the age-standardized results have been analysed according to the Weibull distribution model. The results show that NSM condensate has less than 25% of the potency of tobacco condensate (37% at 95% upper confidence limit), and that condensates from blends of NSM and tobacco are similarly reduced in activity. General pathology analysis failed to reveal abnormalities due to NSM.

Psychosocial reactions of children with cancer. A program for rehabilitation.

The diagnosis of cancer in children challenges all the skills of the pediatric nurse. She utilizes her basic knowledge of development, family dynamics, and disease process. She explores all the implications of long-term illness. She assesses, plans, implements, and evaluates care for the child and his family. She acts as liaison, teacher, and counselor. She is the advocate for the child and his family. As an active member of the team she is instrumental in the process of rehabilitation for the child, physically and emotionally. She can help the child to live with cancer and assure quality of life in the days granted him.