No increase of apoptosis in regressing mouse liver after withdrawal of growth stimuli or food restriction.

In short-term in vivo experiments, liver growth and regression in mice with high (C3H/He), intermediate (B6C3F1) or low (C57BL/6J) susceptibility to hepatocarcinogenesis was compared. Liver growth was induced by dietary administration of phenobarbital (PB; 750 ppm) or nafenopin (NAF; 500 ppm). PB or NAF treatment for 7 days produced moderate increases of liver DNA (15% or 25-28%, respectively) along with pronounced hypertrophy. Liver growth was strongest in C3H/He mice. Cessation of PB or NAF treatment led to a rapid regression of liver hypertrophy. However, the enhanced hepatic DNA content persisted for at least 2 weeks in all mouse strains. Apoptosis was not increased at any time after cessation of treatment in all strains. Food restriction to 60% of the ad libitum intake did not amplify either regression of liver hyperplasia or the occurrence of apoptosis. No strain difference in the occurrence of apoptosis was detected. Mouse hepatocytes in liver regressing after mitogen withdrawal do not enter apoptosis as readily as rat hepatocytes.     

Role of apoptosis for mouse liver growth regulation and tumor promotion: comparative analysis of mice with high (C3H/He) and low (C57Bl/6J) cancer susceptibility

Apoptosis constitutes one of the organisms defense lines against cancer. We investigated whether failure of apoptosis may be concurrently causative for the high cancer susceptibility in C3H/He as compared to C57BL/6J mice (low cancer susceptibility). First, in short-term in vivo experiments (7-21 days), mouse liver growth (C3H/He, C57BL/6J) was induced by administration of phenobarbital (PB; 2 days 500 ppm + 5 days 750 ppm via the food) or nafenopin (NAF; 7 days 500 ppm via the food), cessation of PB or NAF treatment served to initiate liver involution. Liver weight, DNA content, hepatocyte ploidy and apoptotic activity were studied as endpoints. Secondly, in a long-term study liver carcinogenesis was initiated by a single dose of N-nitrosodiethylamine (NDEA, 90 mg/kg b.w.) to 5-weeks-old C57Bl/6J and C3H/He mice. After 2 weeks, mice received either standard diet or a diet containing phenobarbital (PB, 90 mg/kg b.w.) for up to 90 weeks. Cell proliferation and apoptosis in normal liver tissue and (pre)neoplastic tissue was quantitatively analysed by histological means. The short term studies revealed that PB and NAF-induced mouse liver growth is essentially due to cell enlargement (hypertrophy). A moderate increase of liver DNA content was brought about by hepatocellular polyploidization; C3H/He mice exhibited the most pronounced ploidy shift, corresponding to their high cancer susceptibility. Upon cessation of PB or NAF treatment, regression of liver mass was neither associated with a loss of DNA nor an increase in apoptoses in the liver of C3H/He and C57Bl/6J mice; food restriction did not enforce the occurrence of apoptosis. Thus, the mouse strains did not differ with respect to the occurrence of apoptosis. In the long-term study, PB promoted liver tumor formation in all strains, exhibiting quantitative differences in growth kinetics of preneoplasia rather than a specific biological quality. Quantitative analysis of apoptosis in normal and (pre)neoplastic liver tissue of C3H/He and C57BL/6J mice revealed no clue to explain their different cancer susceptibility. Rather, cell proliferation seems to be the prevailing determinant of tumor promotion in the liver of both mouse strains.     

Cytokeratin 8/18 as a new marker of mouse liver preneoplastic lesions

To search for a reliable biomarker of preneoplastic lesions arising early in mouse hepatocarcinogenesis the proteomes of microdissected basophilic foci, hepatocellular adenomas (HCAs), carcinomas (HCCs) and normal-appearing liver of B6C3F1 mice initiated with diethylnitrosamine (DEN) were analysed on anionic (Q10) surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) ProteinChip arrays. Significant overexpression of cytokeratin 8 (CK8; m/z 54, 565), cytokeratin 18 (CK18; m/z 47,538) proteins was found in basophilic foci as well as in HCAs and HCCs. Furthermore, immunohistochemistry demonstrated profound overexpression of CK8 and CK18 proteins (CK8/18) in all basophilic foci, mixed cell type foci, HCAs and HCCs in B6C3F1 and C57BL/6J mice initiated with DEN. A strong correlation between CK8/18-positive foci development and multiplicity of liver tumors in B6C3F1 and C57Bl/6J mice was further observed. Moreover, formation of CK8 and CK18 complexes due to CK8 phosphorylation at Ser73 and Ser431 was found to be strongly associated with neoplastic transformation of mice liver basophilic foci. Elevation of CK8/18 was strongly correlated with induction of cell proliferation in basophilic foci and tumors. In conclusion, our data imply that CK8/18 is a novel reliable marker of preneoplastic lesions arising during mouse hepatocarcinogenesis which might be used for prediction of tumor development and evaluation of environmental agents as well as drugs and food additives using mouse liver tests.     

Increased susceptibility to mouse hepatitis virus 3 of peritoneal macrophages exposed to dieldrin

Interaction of a single dose (36 mg/kg body wt) of the organochlorine pesticide dieldrin with mouse peritoneal macrophages was examined in C57Bl/6, (C57Bl/6 X A/J)F1, and A/J strains of different genetic resistance to mouse hepatitis virus 3 (MHV3) infection. In vivo studies showed increased susceptibility to MHV3 acute disease of C57Bl/6 and (C57Bl/6 X A/J)F1 animals challenged with the pesticide. Significant decrease of mean time of death in dieldrin-exposed, MHV3-infected susceptible C57Bl/6 mice was observed similarly upon po or ip administration of a single, sublethal dose of dieldrin. In addition, decrease of humoral response to the virus was quantified by determination of anti-MHV3 IgG antibodies in spleen cell supernatant fractions and in blood sera of dieldrin-exposed C57Bl/6 mice. A single dose of dieldrin did not alter the in vivo resistance of A/J animals to acute MHV3 disease. The resistant A/J mice, however, showed increased mortality upon two subsequent exposures to dieldrin followed by infection with high lethal doses of MHV3. Phagocytic activity, cell adherence capacity, and attachment and uptake of 3H-radiolabeled MHV3 by C57Bl/6 peritoneal macrophages were determined by in vitro studies. These affector activities of peritoneal macrophages were slightly decreased or unchanged in cells originating from animals exposed to the pesticide. However, the intrinsic activity of MHV3 restriction appeared to be affected in macrophages derived from dieldrin-treated animals: (i) peritoneal C57Bl/6 macrophages collected from the early phase of acute MHV3 disease contained increased MHV3 antigen and (ii) increased cytolysis was observed after in vitro MHV3 infection of macrophages originating from dieldrin-exposed C57Bl/6 mice.     

Sustained induction of hepatic xenobiotic metabolising enzyme activities by phenobarbitone in C3H/He mice: relevance to nodule formation

Phenobarbitone (PB) was administered to male C3H/He mice at a dose of 85 mg/kg/day in a semisynthetic diet for up to 90 weeks. Throughout the treatment period a sustained induction of a number of parameters of hepatic Phase I and Phase II xenobiotic metabolism was observed. Histological examination revealed hypertrophy of the centrilobular cells of the liver lobule in PB treated mice and after 25 weeks small basophilic nodules were found in control and PB treated animals. In addition eosinophilic nodules, which were often large, developed in PB treated mice. Xenobiotic metabolising enzyme activities in large excised nodules after 70 or 90 weeks of PB treatment were either similar to or greater than those present in surrounding host tissue. Both phenobarbitone- and polycyclic hydrocarbon-type mixed function oxidase enzyme activities were induced in large nodules. In conclusion, PB produced a sustained induction of xenobiotic metabolising enzymes both in host tissue and in large eosinophilic nodules. The formation of these nodules in C3H/He mice was thus not associated with any failure of induction of hepatic xenobiotic metabolism.     

Differential susceptibility to acetaminophen-induced liver injury in sub-strains of C57BL/6 mice: 6N versus 6J

Mouse models of acetaminophen (APAP) hepatotoxicity are considered relevant for the human pathophysiology. The C57BL/6 strain is most popular because it is the background strain of gene knock-out mice. However, conflicting results in the literature may have been caused by sub-strain mismatches, e.g. C57BL/6J and C57BL/6N. This study was initiated to determine the mechanism behind the sub-strain susceptibility to APAP toxicity. C57BL/6N and C57BL/6J mice were dosed with 200 mg/kg APAP and sacrificed at different time points. C57BL/6N mice developed significantly more liver injury as measured by plasma ALT activities and histology. Although there was no difference in glutathione depletion or cytochrome P450 activity between groups, C57BL/6N had a higher glutathione disulfide-to-glutathione ratio and more APAP protein adducts. C57BL/6N showed more mitochondrial translocation of phospho-JNK and BAX, and more release of mitochondrial intermembrane proteins apoptosis-inducing factor (AIF), second mitochondria-derived activator of caspases (SMAC), which caused more DNA fragmentation. The increased mitochondrial dysfunction was confirmed in vitro as C57BL/6N hepatocytes had a more precipitous drop in JC-1 fluorescence after APAP exposure. Conclusion: C57BL/6N mice are more susceptible to APAP-induced hepatotoxicity, likely due to increased formation of APAP-protein adducts and a subsequent enhancement of mitochondrial dysfunction associated with aggravated nuclear DNA fragmentation.     

Phenobarbital induces progressive patterns of GC-rich and gene-specific altered DNA methylation in the liver of tumor-prone B6C3F1 mice.

Altered DNA methylation contributes to tumorigenesis by affecting gene expression in a heritable fashion. Phenobarbital (PB) is a nongenotoxic rodent carcinogen which induces global hypomethylation and regions of hypermethylation in mouse liver. Liver tumor-sensitive (B6C3F1) and -resistant (C57BL/6) male mice were administered 0.05% (wt/wt) PB in drinking water for 2 or 4 weeks, and a 2-week recovery was included following each dosing period. DNA was isolated from liver (target) and kidney (nontarget) tissues. The methylation status of GC-rich regions of DNA was assessed via methylation-sensitive restriction digestion, arbitrarily primedpolymerase chain reaction, and capillary electrophoretic separation of products. PB-induced regions of altered methylation (RAMs) which carry forward from an early to a later time point are more likely to be mechanistically relevant as compared to those that do not. Twelve of 69 RAMs (17%) present in B6C3F1 liver at 2 weeks were also seen at 4 weeks, while only 1 of the 123 RAMs (< 1%) present in C57BL/6 liver was seen at 4 weeks. In the B6C3F1 mice, 57 unique (as compared to the C57BL/6) regions of altered hepatic methylation (RAMs), predominantly hypomethylation, were observed at 2 weeks, increasing to 86 at 4 weeks. Changes in methylation were largely reversible. Altered methylation in liver was highly dissimilar to that of kidney. Following 4 weeks PB, bisulfite sequencing revealed hypomethylation of Ha-ras in B6C3F1, but not C57BL/6, which correlated with increased gene expression. These data indicate that (1) progressive, nonrandom changes in methylation provide an epigenetic mechanism underlying the ability of PB to cause mouse liver tumorigenesis and (2) susceptibility to tumorigenesis is related inversely to the capacity to maintain normal patterns of methylation.     

乙醇对嗜酒和非嗜酒小鼠体重和肝脏组织形态的影响

目的比较乙醇对嗜酒和非嗜酒小鼠躯体损害的易感性。方法以近交系C57BL/6J小鼠(嗜酒者)和远交系ICR小鼠(非嗜酒者)为实验动物,采用强制性饮酒程序:饮酒组小鼠饮用乙醇溶液8d,乙醇浓度从3%递增至20%。每天检测小鼠体重、摄食量、饮液量和乙醇摄入量。饮用乙醇期间取小鼠肝脏用组织化学方法观察肝脏组织病理学变化。结果乙醇对嗜酒小鼠体重增长的抑制作用明显强于非嗜酒小鼠。乙醇对两组小鼠的摄食量均有抑制作用,抑制程度无显著差异。乙醇抑制非嗜酒小鼠的饮液量,但对嗜酒小鼠无影响。实验期间两组小鼠的饮酒量相当,但嗜酒小鼠的肝脏损伤更为严重。结论嗜酒小鼠对乙醇毒性作用的耐受性弱于非嗜酒小鼠。     

Comparison of tissue dosimetry in the mouse following chronic exposure to arsenic compounds

Several chronic bioassays have been conducted in multiple strains of mice in which various concentrations of arsenate or arsenite were administered in the drinking water without a tumorigenic effect. However, one study (Ng et al., 1999) reported a significant increase in tumor incidence in C57Bl/6J mice exposed to arsenic in their drinking water throughout their lifetime, with no tumors reported in controls. A physiologically based pharmacokinetic model for arsenic in the mouse has previously been developed (Gentry et al., 2004) to investigate potential differences in tissue dosimetry of arsenic species across various strains of mice. Initial results indicated no significant differences in blood, liver, or urine dosimetry in B6C3F1 and C57Bl/6 mice for acute or subchronic exposure. The current work was conducted to compare model-predicted estimates of tissue dosimetry to additional kinetic information from the (C57Bl/6 xCBA)F1 and TgAc mouse. The results from the current modeling indicate that the pharmacokinetic parameters derived based on information in the B6C3F1 mouse adequately describe the measured concentrations in the blood/plasma, liver, and urine of both the (C57Bl/6 x CBA)F1 and TgAc mouse, providing further support that the differences in response observed in the chronic bioassays are not related to strain-specific differences in pharmacokinetics. One significant finding was that no increases in skin or lung concentrations of arsenic species in the (C57Bl/6 x CBA)F1 strain were observed following administration of low concentrations (0.2 or 2 mg/U of arsenate in the drinking water, even though differences in response in the skin were reported. These data suggest that pharmacodynamic changes may be observed following exposure to arsenic compounds without an observable change in tissue dosimetry. These results provided further indirect support for the existence of inducible arsenic efflux in these tissues.     

Inhibition of morphine-induced analgesia and locomotor activity in strains of mice: a comparison of long-acting opiate antagonists

The long-acting opiate antagonistic potency of naloxazone (NXZ), beta-chlornaltrexamine (beta-CNA) and beta-funaltrexamine (beta-FNA) was compared using three inbred strains of mice, in which morphine induces either analgesia (DBA/2), locomotion (C57BL/6), or both responses (C3H/He). The antagonists were applied SC 24-120 hr before morphine (10 or 20 mg/kg, IP), followed by the tests after 30 min. The minimal dose which completely antagonized morphine-induced analgesia in DBA and locomotion in C57 mice during 24 hr were: for NXZ 50 and 100 mg/kg, for beta-CNA 0.8 and 6.2 mg/kg, for beta-FNA 1.6 and 12.5 mg/kg, respectively. beta-FNA and beta-CNA more potently blocked morphine-induced analgesia in DBA mice than the activity response in the C57 strain. In contrast, beta-FNA prevented morphine-induced locomotion at a lower dose (6.2 mg/kg) than analgesia (greater than 50 mg/kg) in C3H mice, while beta-CNA was equipotent (1.6 mg/kg). In general, beta-CNA turned out to be the most reactive compound, antagonizing morphine effects in low doses up to 120 hr. beta-FNA selectively antagonized either morphine-induced analgesia or locomotion, depending on the strain used. This suggests that a given morphine response might be caused by a genetically determined multiplicity of opiate receptor types and their mutual interactions.     

A Wistar rat strain prone to spontaneous liver tumor development: implications for carcinogenic risk assessment

The European Union legislation considers nongenotoxic substances that only cause liver tumors in certain sensitive strains of mice as raising no concern for man. The EU legislation, however, also clearly stipulates that cases where the only available tumor data are the occurrence of neoplasms at sites and in strains where they are well known to occur spontaneously with a high incidence are relevant arguments which exclude a concern for man. We have analyzed the spontaneous liver tumor incidence in Wistar rats and in B6C3F(1) and C57Bl mice used in carcinogenicity trials at the BASF facility in Ludwigshafen, Germany, over the past 15 years and compared the spontaneous liver tumor incidence in BASF Wistar rats with those observed in rat strains employed in major European contract research organizations (CROs). The results of these analyses indicate that the incidence of spontaneous liver tumors in the BASF Wistar rat strain is very high, similar to that seen in the B6C3F(1) mouse and much higher than that seen in the C57Bl mouse and other commonly used strains of rat. The analyses also revealed signs of increasing variability and liver tumor drift in several rat strains. Moreover, the incidence of spontaneous preneoplastic liver cell foci was far higher in the BASF Wistar rat strain than reported for other rat strains in the literature. The analyses provide relevant arguments which exclude a concern for man for nongenotoxic chemicals that only tested positive for liver tumors in this sensitive rat strain.     

Comparison of the acute toxicity of clioquinol, histamine, and chloroform in different strains of mice.

The oral LD50'S of clioquinol, histamine and chloroform and the intravenous LD50 of histamine were determined separately in male and female mice of the Tif : MAGf (SPF), Tif : MF2f (SPF), C3H/Tif Bomf, DBA2/J Bomf, C57Bl/6J/Bomf and A/J Bomf strains. Mice of the Tif : MAGf (SPF) outbred strain and the Tif : MF2f(SPF) hybrids tended to be more resistant than the inbred strains, with exception of C57Bl/6J/Bomf, which proved least susceptible to the lethal effects of the tested preparations. The toxicity of chloroform was more pronounced in the males than in the females, and C3H/Tif Bomf proved more susceptible than all other strains. The toxicity of clioquinol and histamine was not related to sex.     

Limitations on the use of the C57BL/6 mouse in the tail suspension test

RATIONALE: The C57BL/6 is one of the most widely used mouse strains in behavioral, pharmacological, and genetic research but little is known about their response on tests for antidepressant drugs. OBJECTIVES: The behavior of C57BL/6 mice, and mice from other strains, was examined in the tail suspension test (TST), a common behavioral test used for the screening of antidepressant compounds. METHODS: C57BL/6J mice from the Jackson Laboratory, C57BL/6N mice from Harlan, A/J, 129-SV-ter and DBA/2 mice were tested under baseline conditions in the TST. RESULTS: The majority of the C57BL/6 mice from the Jackson Laboratory tested in this paradigm (70%) climbed up their tails during the 6-min test session. C57BL/6 mice obtained from Harlan (35%) also demonstrated this climbing behavior, suggesting that it is not specific to mice from a particular supplier. Other strains (A/J 18%), 129-SV-ter (0%) and DBA/2 (0%) mice) showed less propensity for tail climbing. CONCLUSIONS: The occurrence of this behavior is an important consideration when testing antidepressant drugs or the effects of stress using the TST with inbred mouse strains, especially those from the C57BL/6 strain.     

Effect of chronic phenobarbitone administration on liver tumour formation in the C57BL/10J mouse

The hepatocarcinogenicity of sodium phenobarbitone (PB) was studied in male and female mice of the low spontaneous liver tumour incidence C57BL/10 J strain. Treatment with 200 and 1000 ppm PB for 1 month increased relative liver weight in both sexes, with 1000 ppm PB also producing a transient increase in replicative DNA synthesis. The treatment of male and female mice with 200 and 1000 ppm (the maximum tolerated dose) PB for 99 weeks resulted in centrilobular hypertrophy and a dose-dependent increase in relative liver weight. Altered hepatic foci were observed in both sexes given 1000 ppm PB. In male mice given 1000 ppm PB significant increases were observed in the incidence of hepatocellular adenoma and carcinoma, to 43% and 10% of the animals examined, respectively. No increase in liver tumours was observed in male mice given 200 ppm PB and in female mice given 200 and 1000 ppm PB. In summary, PB at a dose level which produces liver hypertrophy, a transient stimulation of replicative DNA synthesis and on chronic administration altered hepatic foci, three key events in the established mode of action for PB-induced rodent liver tumour formation, results in a significant increase in liver tumours in male C57BL/10 J mice.     

Elevated binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3-methylcholanthrene to the Ah receptor in hepatic cytosols from phenobarbital-treated rats and mice

Binding of 3-methylcholanthrene (MC),2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and other “MC-type” inducers to cytosolic Ah receptor sites is the first specific step in induction of aryl hydrocarbon hydroxylase (AHH; cytochrome P₁?450) by these compounds. [₃H]TCDD and [₃H]MC were used as radioligands to quantitate and characterize Ah receptor in hepatic cytosols from genetically “responsive” C57BL/6J mice, genetically “nonresponsive” DBA/2J mice, and AHH-inducible Sprague-Dawley rats. Injection of 50–100 mg/kg of phenobarbital (PB) for 3 days more than doubled the concentration of Ah receptor in hepatic cytosol from Sprague-Dawley rats. In C57BL/6J mice, PB injection at 25 mg/kg × 3 days significantly increased (P < 0.01) the Ah receptor concentration in hepatic cytosol. No cytosolic Ah receptor was detectable in hepatic cytosol from untreated DBA/2J mice, nor did any Ah receptor appear after PB treatment in this “nonresponsive” strain. Although PB significantly elevated Ah receptor in hepatic cytosols of responsive rodents, many previous studies have shown that the maximal level of AHH activity in animals given PB and an “MC-type” inducer simultaneously is additive rather than synergistic. Ah receptor concentrations can be doubled by PB treatment without doubling the subsequent AHH-induction response to “MC-type” compounds. Thus, the cytosolic Ah receptor concentration per se may not be the primary determinant of a given tissue's maximal capacity for AHH induction by “MC-type” compounds.     

Phenobarbitone-induced liver response in wild type and in p53 deficient mice.

The tumour suppressor protein, p53, is involved in the regulation of apoptosis and growth arrest following DNA damage. Mutations of the p53 gene are found in 50-55% of all human cancers (Hollstein et al. Nucl. Acid Res. 22 (1994) 3551), including hepatocellular carcinomas. Phenobarbitone (PB) is a non-genotoxic hepatocarcinogen in rats and mice. With commercial availability of mice where one or both alleles of p53 have been removed we have examined the effect of PB in wild type C57BL/6J mice (p53 +/+), and p53 deficient mice (+/- and -/- p53) to determine whether p53 plays a role in the PB induced liver response. In each strain of mice, chronic administration caused liver enlargement, which was associated with centrilobular hepatocyte hypertrophy and a transient hyperplasia. In addition, an increase in centrilobular epidermal growth factor receptor and its ligand, transforming growth factor alpha and a decrease in mannose-6-phosphate receptor and its mitoinhibitory ligand, TGFbeta1 was also observed immunohistochemically. The similar response in all three strains indicates that p53 probably plays no role in the early PB induced liver effects of hypertrophy and changes in growth factor expression.     

Liver preneoplastic changes in mice treated with the herbicide fomesafen.

1. Effect of the diphenyl ether herbicide fomesafen on liver preneoplastic changes and porphyrin biosynthesis was examined in male C57BL/6J mice (0.23% in the diet for 14 months) and ICR mice (0.3% in the diet for 50 weeks). Fomesafen treatment resulted in preneoplastic changes (liver nodules and foci of altered hepatocytes) in both strains, uroporphyria developed only in ICR mice. 2. Iron pretreatment (600 mg/kg as a single dose) accelerated the development of fomesafen-induced preneoplastic changes in both mouse strains. The number of foci containing altered hepatocytes, as well as the number and size of liver nodules, were increased in iron-pretreated animals. 3. A single injection of iron induced marked uroporphyria in C57BL/6J mice after 14 months (liver porphyrin content 102 nmol/g). This uroporphyria was further potentiated by fomesafen administration (208 nmol/g). 4. In ICR mice, liver histology was apparently normal after a 3 month recovery from fomesafen treatment (0.32% for 9 months). Liver porphyrin content (260 nmol/g) started to decrease immediately after fomesafen withdrawal, but was still significantly elevated after 3 months (5 nmol/g), as compared to controls (1 nmol/g). 5. It is concluded that the toxicological evaluation of fomesafen should focus on liver porphyrin biosynthesis.     

Experimental envenoming of mice with venom from the scorpion Centruroides limpidus limpidus: differences in mortality and symptoms with and without antibody therapy relating to differences in age, sex and strain of mouse

C57Bl/6J and BALB/cAnN inbred strains of mice differed significantly in mortality and symptoms when intoxicated subcutaneously with one LD₅₀ of venom from Centruroides limpidus limpidus. Higher mortality was observed in C57Bl/6J than in BALB/cAnN. Also, C57Bl/6J mice more quickly developed muscular and respiratory collapse whilst BALB/cAnN mice were hyperactive before dying. Also, the symptoms in the survivors lasted for 24 h in C57Bl/6J and for 2 h in BALB/cAnN. The age and sex of mice were also related to mortality: younger mice were more resistant than older mice and females were more susceptible than males, especially in the younger groups. Antivenom (horse F(ab′)₂) administration 5–10 min after envenoming of mice with one LD₅₀ rescued 60% of BALB/cAnN and 52% of C57Bl/6J mice, respectively. Results indicate that genetic background, gender and age differences are of consequence in the pathogenesis of C. limpidus scorpion envenomation in mice, and that timely treatment with active antivenom F(ab′)₂ saves a significant fraction of intoxicated mice without statistically significant distinction of strains.