Mouse assays

Some Chemical Considerations Relevant to the Mouse Bioassay. Net toxicity, determined by mouse bioassay, has served as a traditional measure of toxin quantity and, despite the development of HPLC and other detection methods for the saxi-toxins, continues to be used. In this assay, as in most others, the molar specific potencies of the various saxitoxins differ, thus, net toxicity of a toxin sample with an undefined mixture of the saxitoxins can provide only a rough approximation of the net molar concentration. Still, to the extent that limits can be placed on variation in toxin composition, the mouse assay can in principle provide useful data on trends in net toxin concentration. However, the somewhat protean chemistry of the saxitoxins makes it difficult to define conditions under which the composition of a mixture of toxins will remain constant thus, attaining a reproducible level of mouse bioassay toxicity is difficult. It is therefore useful to review briefly some of the chemical factors that should be considered when employing the mouse bioassay for the saxitoxins or when interpreting results. Similar concepts will apply to other assays. 

Figure 9. Observed increases in mouse assay toxicity of test mixtures of shellfish meat and toxin Cl (4), hydrolyzed with varying concentrations of HCI acid. Two series of experiments are shown. The initial concentration of toxin Cl was uniform for all samples in a series. Toxicity is expressed on the vertical axis as percentage of the maximum toxicity attained for that series.

Assays of ciguatoxin. Determination of ciguatoxin levels in fish was carried out in many laboratories by mouse assays. Enzyme immunoassay to screen inedible fish has been proposed by Hokama (9). No specific chemical assay has been developed, as information on functional groups suitable for fluorescence labeling is not available. Analyses conducted in the authors laboratory on remnant fish retrieved from patients meals indicated that ciguatoxin content as low level as 1 ppb could cause intoxication in adults. An extremely high sensitivity and a sophisticated pretreatment method will be required for designing a fluorometric determination method for the toxin. 

In view of the activity of 13, but the lack of activity for the alpha-ethyl homolog of DOM, the two isomeric ring-methylated derivatives Structures 16a and 16b were recently prepared (114). Neither isomer showed significant activity, either as an agonist in the rat fundus preparation or in a mouse assay, when compared with 13. It would appear that little bulk can be tolerated near the alpha-carbon, other than a methyl or methylene. 

Chronic inhalation exposure of rodents to 1,2-dibromoethane has been associated with neoplasms in the respiratory tract, as well as in other organ systems. Two studies have examined the carcinogenic potential of 1,2-dibromoethane in rodents after inhalation exposure (NTP 1982 Wong et al. 1982). There was also an A strain mouse assay (Adkins et al. 1986). 

Macdonald J, Freanch JE, Gerson RJ, et al. The utility of genetically modified mouse assays for identifying human carcinogens a basic understanding and path forward. Toxicol Sci 2004 77 188-94. 

The early work of Sommer and associates established a good mouse assay for the poison in shellfish products which made quantitative work with the poison practical. A mouse unit (NU) was defined as the minimum amount of poison that would kill a 20-gram white mouse in 15 minutes when one ml of an extract of shellfish was injected intraperitoneally. Higher amounts than the minimum kill in shorter time, i.e., death times of 3, 4, 6, and 8 minutes are equivalent to 3.7, 2.5, 1.6, and 1.3 MU, respectively, as illustrated in Figure 1. 

Also, the reference standard made possible the reporting of the poison content of shellfish as pg per 100 grams of meats and established a basis for an official AOAC mouse assay for the poison (12.13). The Food and Drug Administration set a top limit of 80 micrograms of poison per 100 grams of shellfish meats as marketable for human consumption. This limit is well below the 1 to 4 mg deemed necessary in foods to cause sickness and death of humans. 

As mentioned above we obtained most of the poison for our chemical and biological work from the Alaska butter clam. However our search for a dinoflagellate or any other poisonous organism in the water as a source of the poison in the clams was never definitely accomplished. In 1948, and in some years following, along with a survey for toxic butter clams in Southeastern Alaska by the staff at the Fishery Products Research Laboratory at Ketchikan, 1 collected plankton in areas where the clams were found to be toxic. Mouse assays of the plankton showed no toxicity. Microscopic examination of the water in these areas showed heavy growth of microscopic plankton but little or no evidence of... 

In an assay that offers acceptable HOPi/ri ratios for the carbamates, potential HOP can be estimated by preparing the sample under conditions that insure hydrolysis of the sulfamates. Unfortunately, the conditions specified for sample preparation in the standard mouse bioassay are not sufficiently acidic to insure complete hydrolysis ( ). As currently employed in state monitoring laboratories, the mouse assay may substantially underestimate the potential HOP of samples containing the sulfamate toxins. 

Toxins were extracted with 0.1 N HCl or 75% EtOH acidified to pH 2 and treated with successive columns of charcoal, Bio-Gel P-2 and Bio-Rex 70. Toxins separated by the last column were identified by tic and electrophoresis. Relative abundance of each toxin was determined by monitoring the eluate from Bio-Rex 70 column by mouse assay. A fluorometric paralytic shellfish toxin analyzer was applied to samples which were too small to be followed by mouse assay. Toxins separated by the ion exchange column (Hitachi gel 3011C) were continuously aromatized by tei t-butylhydroperoxide and monitored by the fluorometer (1 ). 

The current test for the shellfish toxins is based on a time-of-death mouse assay (J ). This test is laborious to perform and relatively expensive (15). Consequently, a variety of assay... 

Mouse Assays. Outbred female Swiss mice (Harlan Sprague Dawley ICR BR ) weighing 19 to 21 g were used. Doses of toxic extract were suspended in 0.5 ml of 0.1% Tween 60 in 0.15 M NaCI and administered by i.p. injection. Mice were observed for a period of 48 hours. 

A recent area of major concern has been the development of a practical and specific assay for the detection of ciguatoxin directly from fish tissues. Earlier assays, some of which are still used, relied on whole tissues, crude extracts and partially purified ciguatoxin (8, 9, 20). For the mongoose and cat assays, large amounts (10-15% of body weight) of fresh tissues were required, while the mouse assay required concentrated lipid extracts of the fish tissue. 

TTX has so far been determined mainly by mouse assay ( 4). This assay is featured by simplicity, but has some demerits such as low sensitivity, low accuracy, rather high cost of suitable mice, and local... 

Under these circumstances, attempts have been made to develop chemical assay methods of TTX. In the near future, the mouse assay method may be replaced by chemical methods. 

Tarczynska, M., Nalecz-Jawecki, G., Brzychcy, B., Zalewski, M. and Sawicki, J. (2000) The toxicity of cyanobacterial blooms as determined by microbiotests and mouse assays, in G. Persoone, C. Janssen and W.M. De Coen (eds.), New Microbiotests for Routine Toxicity Screening and Biomonitoring, Kluwer Academic/Plenum Publishers, New York, pp. 527-532. 

Bouwknecht, J. A. and Paylor, R. (2002) Behavioral and physiological mouse assays for anxiety a survey in nine mouse strains. Behav Brain Res 136, 489-501. 

Flammang TJ, Von Tungeln LS, Kadlubar FF, Fu PP. Neonatal mouse assay for tumorigenicity alternative to the chronic rodent bioassay. Regul Toxicol Pharmcol 1997 26 230 10. 

One of the greatest concerns about using undifferentiated stem cells is their potential to develop tumors [67,68]. The investigation of immortalization, malignant transformation, and tumorigenicity, however, are unlikely to be addressed by conventional rodent bioassays for carcinogenicity or the alternative short-term transgenic mouse assays (see Table 33.7). 

COC (2002) Committee on Carcinogenicity of Chemicals in Food, Consumer Products and the Environment (COC). COC statement on ILSI/HESI research programme on alternative cancer models. COC/02/S3, April 2002 Flammang TJ, Von Tungeln LS, Kadlubar FF, Fu PP (1997) Neonatal mouse assay for tumorigenicity. Alternative to the Chronic rodent bioassay. Reg Toxicol Pharmacol 26 230-240... 

Anti-HIV-1 Activity of Calanolides in Hollow Fiber Mouse Evaluation of (-l-)-calanolide A (1) in a hollow fiber culture-based in a SCID mouse assay of antiviral efficacy indicated that (+)-calanolide A exhibited significant anti-HIV-1 activity after oral or parenteral administration on a once-daily (200mg/kg/ dose) or twice-daily (150 mg/kg/dose) treatment. Furthermore, a synergistic effect was observed in the combination of (-l-)-calanolide A and AZT. ... 

Yasumoto, T. 2001. Suitability of two mouse assay methods to quantify marine toxins in bivalve molluscs. Submission to EU Reference Laboratory, Vigo, Spain. 

In a study of anticonvulsant agents, the (S)-benzene ring analog (90) was somewhat more potent in a mouse assay than was the (S)-cyclohexane analog (91) (56). There was... 

Eastin WC, Mennear JH, Tennant R, et al. Tg.AC genetically altered mouse Assay working group overview of available data. Toxicol Pathol. 2001 29S 60-80. 

Unfortunately, the dose-survival times for the DSP toxins in the mouse assay fluctuate considerably and fatty acids interfere with the assay, giving false-positive results consequently, a suckling mouse assay that has been developed and used for control of DSP measures fluid accumulation after injection of the shellfish extract. Considerable effort has been applied recently to development of chemical assays to replace these bioassays. As a result a good high performance liquid chromatography (HPLC) procedure has been developed to identify individual PSP toxins (detection limit for saxitoxin = 20 fg per 100 g of meats 0.2 ppm), an excellent HPLC procedure (detection limit for okadaic acid = 400 ngg 0.4 ppm), a commercially available immunoassay (detection limit for okadaic acid=lfg per 100 g of meats 0.01 ppm) for DSP, and a totally satisfactory HPLC procedure for ASP (detection limit for domoic acid = 750 ngg 0.75 ppm). 

Busby WF Jr, Stevens EK, Martin CN, et al. 1989. Compartive lung tumorigenicity of parent and mononitro-polynuclear aromatic hydrocarbons in the BLU Ha newborn mouse assay. Toxicol Appl Pharmacol 99(3) 555-563. 

Wislocki PG, Bagan ES, Lu AY, et al. 1986. Tumorigenicity of nitrated derivatives of pyrene, benz[a]anthracene, chrysene and benzo[a]pyrene in the newborn mouse assay. Carcinogenesis 7 1317-1322. 

Jet A fuel induced gene mutation in mouse cells in presence of metabolic activation (mouse or rat liver S9) straight-run kerosene positive in mouse assay in presence of metabolic activation JP-4 not mutagenic in mouse assay MD API 81-07 not mutagenic in mouse assay, did not induce SCEs in Chinese hamster ovary cells... 

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