Absence of developmental toxicity in a canine model after infusion of a hemoglobin-based oxygen carrier: Implications for risk assessment.

Bovine-derived hemoglobin-based oxygen carriers (HBOCs) have been investigated for use in humans (HBOC-201) and approved for veterinary medicine (HBOC-301). We infused pregnant beagles with HBOC-201 to test whether HBOC-induced developmental toxicity previously observed in rats would occur in a species devoid of an inverted visceral yolk sac (invVYS). Phase 1 assessed developmental toxicity of 6g/kg HBOC-201 on gestational day (GD) 21. Phase 2 investigated single infusions of 6g/kg HBOC-201 on one of GDs 21, 25, 29 or 33. Phase 3 studied multiple sequential infusions on GDs 21, 23,25,27,29, 31, and 33 at 0.52g/kg/day (3.6g/kg total dose). Mild to moderate maternal toxicity occurred in all phases. There was an unequivocal absence of developmental toxicity in all phases. Overall, our hypothesis that HBOC, which interferes with the function of the invVYS, would not affect the offspring in dogs was supported. The implications relative to human risk are discussed.

Developmental toxicity in rats of a hemoglobin-based oxygen carrier results from impeded function of the inverted visceral yolk sac.

HBOC-201 is a bovine-derived, cross-linked, and stabilized hemoglobin (250kDa) in physiological saline. Daily intravenous infusions of HBOC (1.95, 3.90, or 5.85g/kg/day) during gestational days (GDs) 6-18 in Sprague-Dawley rats caused fetal mortality, reduced birth weight, and malformations. Subsequent single-day infusions (5.85g/kg/day) showed that developmental toxicity was limited to GDs 7-9 when histiotrophic nutrition via the inverted visceral yolk sac (invVYS) is essential. Histiotrophic nutrition is receptor-mediated endocytosis of bulk maternal proteins and subsequent lysosomal degradation providing amino acids and other nutrients for embryonic growth. Controls for protein content, oncotic properties, and hemoglobin content indicated that toxicity was due to hemoglobin. Rat whole embryo cultures verified HBOC interference with invVYS transport capacity and resultant deficient embryonic nutrition. These mechanisms of action are not expected to impact human development based on differences in VYS morphology and function, although a complete understanding of early human embryonic nutrition is lacking.

The cDNA encoding Xenopus laevis heat-shock factor 1 (XHSF1): nucleotide and deduced amino-acid sequences, and properties of the encoded protein.

We have isolated a cDNA encoding Xenopus laevis (Xl) heat-shock factor 1 (XHSF1). XHSF1, translated from the mRNA synthesized in vitro, will bind specifically to the Xl hsp70 promoter (hsp70). Microinjection of XHSF1 mRNA into Xl oocytes leads to synthesis of XHSF1 which accumulates in the nucleus and selectively activates Xl phsp70p activity at 18 degrees C.

Absence of prenatal developmental toxicity from inhaled arsenic trioxide in rats.

A review of the literature revealed no published inhalational developmental toxicity studies of arsenic performed according to modern regulatory guidelines and with exposure throughout gestation. In the present study, inorganic arsenic, as arsenic trioxide (As(+3), As2O3), was administered via whole-body inhalational exposure to groups of twenty-five Crl:CD(SD)BR female rats for six h per day every day, beginning fourteen days prior to mating and continuing throughout mating and gestation. Exposures were begun prior to mating in order to achieve a biological steady state of As(+3) in the dams prior to embryonal-fetal development. In a preliminary exposure range-finding study, half of the females that had been exposed to arsenic trioxide at 25 mg/m3 died or were euthanized in extremis. In the definitive study, target exposure levels were 0.3, 3.0, and 10.0 mg/m3. Maternal toxicity, which was determined by the occurrence of rales, a decrease in net body weight gain, and a decrease in food intake during pre-mating and gestational exposure, was observed only at the 10 mg/m3 exposure level. Intrauterine parameters (mean numbers of corpora lutea, implantation sites, resorptions and viable fetuses, and mean fetal weights) were unaffected by treatment. No treatment-related malformations or developmental variations were noted at any exposure level. The no-observed-adverse-effect level (NOAEL) for maternal toxicity was 3.0 mg/m3; the NOAEL for developmental toxicity was greater than or equal to 10 mg/m3, 760 times both the time-weighted average threshold limit value (TLV) and the permissible exposure limit (PEL) for humans. Based on the results of this study, we conclude that arsenic trioxide, when administered via whole-body inhalation to pregnant rats, is not a developmental toxicant.

Interpreting the toxicologic significance of alterations in anogenital distance: potential for confounding effects of progeny body weights.

Anogenital distance (AGD) is an endpoint that was recently added to the U.S. EPA testing guidelines for reproductive toxicity studies. This endpoint is sensitive to hormonal effects of test chemicals. It is possible that apparent alterations in AGD might occur after treatment with agents that affect overall pup body size. In such cases, hormonal activity might be associated incorrectly with the test treatment. The analyses in this report evaluated statistical correlations between pup body weight and AGD in control litters. AGDs were measured on postnatal day 1 in 1501 pups derived from 113 untreated female Sprague-Dawley rats in two independent two-generation reproductive toxicity studies. Significant correlations were detected between AGD and body weight and between AGD and the cube root of body weight. In males, AGD increased 0.26 mm for each 1 g increase in body weight. In females, AGD increased 0.13 mm per 1 g increase in body weight. Although there were essentially no differences between the regression models developed to predict AGD in either males or females using body weight as a covariate and those based on the cube root of body weight, such similarities in predictivity might not occur in larger animals with broader weight ranges than those encountered in this analysis. Normalization of AGD by dividing by body weight significantly overcompensated for differences in body size. Normalizing with the cube root of body weight resulted in an AGD/cube root of body weight ratio that was constant across the range of body weights observed in this study. In conclusion, as a preferred method to account for body size effects on AGD, analysis of covariance is recommended. If a normalization is done directly, the ratio of AGD to the cube root of body weight is the more appropriate metric.

Evaluation of the prenatal developmental toxicity of orally administered arsenic trioxide in rats.

A thorough review of the literature revealed no published repeated-dose oral developmental toxicity studies of inorganic arsenic in rats. In the present study, which was conducted according to modern regulatory guidelines, arsenic trioxide was administered orally beginning 14 days prior to mating and continuing through mating and gestation until gestational day 19. Exposures began prior to mating in an attempt to achieve a steady state of arsenic in the bloodstream of dams prior to embryo-foetal development. Groups of 25 Crl:CD(SD)BR female rats received doses of 0, 1, 2.5, 5 or 10mg/kg/day by gavage. The selection of these dose levels was based on a preliminary range-finding study, in which excessive post-implantation loss and markedly decreased foetal weight occurred at doses of 15 mg/kg/day and maternal deaths occurred at higher doses. Maternal toxicity in the 10mg/kg/day group was evidenced by decreased food consumption and decreased net body weight gain during gestation, increased liver and kidney weights, and stomach abnormalities (adhesions and eroded areas). Transient decreases in food consumption in the 5mg/kg/day group caused the maternal no-observed-adverse-effect level (NOAEL) to be determined as 2. 5mg/kg/day. Intrauterine parameters were unaffected by arsenic trioxide. No treatment-related foetal malformations were noted in any dose group. Increased skeletal variations at 10mg/kg/day were attributed to reduced foetal weight at that dose level. The developmental NOAEL was thus 5mg/kg/day. Based on this study, orally administered arsenic trioxide cannot be considered to be a selective developmental toxicant (i.e. it is not more toxic to the conceptus than to the maternal organism), nor does it exhibit any propensity to cause neural tube defects, even at maternally toxic dose levels.

Developmental toxicity evaluation of a scrubbing solution used in petroleum refineries.

The developmental toxicity potential of a scrubbing solution used extensively in petroleum refineries to remove CO2 from hydrogen gas streams was evaluated via inhalation. Pregnant female CD (Sprague-Dawley) rats were exposed to aerosols of a "used" scrubbing solution at 0.05, 0.1, 0.2, or 0.3 mg/l for 6 h/d on d 6-19 of pregnancy. Control animals were exposed to filtered air under the same exposure conditions. Dams were sacrificed on d 20 of pregnancy and a laparohysterectomy was performed. The mass median aerodynamic diameter of the aerosol revealed that all particles ranged from 1.6 to 2.8 microm, with geometric standard deviations between 2.0 and 2.3 microm. The overall pregnancy rate was high (>95%) and equivalent across all groups. All pregnant dams had live litters, and 22-24 litters were examined in each group. Treatment-related clinical signs consisted of rales, observed at all exposure levels, and gasping noted only at the 0.3 mg/l exposure level. The occurrence of rales was presumably a localized effect on the respiratory tract and likely due to the irritating properties of the scrubbing solution. Maternal toxicity was exhibited in the 0.3 mg/l group, including reduced body weight, weight gain, and food consumption and one possible treatment-related death on gestation d 17. At scheduled necropsy, there were no treatment-related gross pathological observations and no statistically significant reproductive and developmental effects. The incidences of fetuses with skeletal variations involving the sternum were clustered in two litters at the highest exposure level with atypically low term fetal body weights. Under the conditions of this investigation, potassium carbonate scrubbing solution is not a selective developmental toxicant.

Site-directed mutagenesis of the T4 endonuclease V gene: role of tyrosine-129 and -131 in pyrimidine dimer-specific binding.

T4 endonuclease V incises DNA at the sites of pyrimidine dimers through a two-step mechanism. These breakage reactions are preceded by the scanning of nontarget DNA and binding to pyrimidine dimers. In analogy to the synthetic tripeptides Lys-Trp-Lys and Lys-Tyr-Lys, which have been shown to be capable of producing single-strand scissions in DNA containing apurinic sites, endonuclease V has the amino acid sequence Trp-Tyr-Lys-Tyr-Tyr (128-132). Site-directed mutagenesis of the endonuclease V gene, denV, was performed at the Tyr-129 and at the Tyr-129 and Tyr-131 positions in order to convert the Tyr residues to nonaromatic amino acids to test their role in dimer-specific binding. The UV survival of repair-deficient (uvrA recA) Escherichia coli cells harboring the denV N-129 construction was dramatically reduced relative to wild-type denV+ cells. The survival of denV N-129,131 cells was indistinguishable from that of the parental strain lacking the denV gene. The mutant endonuclease V proteins were then characterized with regard to (1) dimer-specific nicking activity, (2) apurinic nicking activity, and (3) binding affinity to UV-irradiated DNA. Dimer-specific nicking activity and dimer-specific binding for both denV N-129 and N-129,131 were abolished, while apurinic-specific nicking was substantially retained in denV N-129,131 but was abolished in denV N-129. These results indicate that Tyr-129 and Tyr-131 positions of endonuclease V are at least important in pyrimidine dimer-specific binding and possibly nicking activity.

Site-directed mutagenesis of rat cellular retinol-binding protein. Alteration in binding specificity resulting from mutation of glutamine 108 to arginine.

Cellular retinol-binding protein (CRBP) is a retinol-specific binding protein. A rat cDNA clone of CRBP was expressed in Escherichia coli. In order to determine amino acid residues in CRBP which may be important for the binding of all-trans-retinol, comparative model-building studies were performed in which strong sequence similarities were identified between CRBP and several other binding proteins. Based on this analysis, specific amino acids were predicted to be important in retinol binding, and these predictions were tested using the technique of site-directed mutagenesis to subtly alter the protein's structure and function. Specifically, site-directed mutagenesis was performed to alter the Gln-108 to Arg-108 (Q108R). Making use of fluorescence, Q108R was found to have a 3-fold lower affinity for all-trans-retinol, and the fine structure of the excitation spectrum of the Q108R.all-trans-retinol complex was also different than for the wild type.all-trans-retinol complex. The mutant bound 13-cis-retinol with an excitation spectrum identical to wild type bound to 13-cis-retinol, but with only one-half of the fluorescence intensity. In competition binding experiments, the Q108R mutant was found to have similar binding affinities for all-trans-retinol, all-trans-retinoic acid, 13-cis-retinoic acid, and retinal, while wild type CRBP was only able to bind to all-trans-retinol. Thus, altering a single amino acid in CRBP (Gln-108 to Arg-108) caused a significant change in the ligand binding specificity of the protein.

A retinoic acid response element from the rat CRBPI promoter is activated by an RAR/RXR heterodimer.

The expression of the rat cellular retinol binding protein I (rCRBPI) can be upregulated in vivo by retinoic acid (RA). Here we have analyzed the rCRBPI promoter region and compared it to the corresponding mouse sequence. We find that the CRBPI 5' flanking region has been highly conserved between rat and mouse, including a RA response element (RARE) approximately 1 kb upstream of the start of transcription. The RARE is of the direct repeat type with a two nucleotide spacer. Like other direct repeat RAREs, this response element is activated by RAR alpha and beta but not by RAR gamma 1. Furthermore, the rCRBPI-RARE is most effectively activated when both RAR and RXR are present. In addition RAR/RXR heterodimers are required for efficient binding to the rCRBPI-RARE, while RARs or RXR alone do not interact effectively with this response element. The rCRBPI gene is therefore most likely activated in vitro by a RAR/RXR heterodimer.

Cloning and expression of a pharmacologically unique bovine peripheral-type benzodiazepine receptor isoquinoline binding protein.

High affinity binding of isoquinolines, such as PK 11195, is a conserved feature of peripheral-type benzodiazepine receptors (PBR) across species. However, species differences in PBR ligand binding have been described based on the affinity for N1-alkyl-1,4-benzodiazepines, such as Ro5-4864. Ro5-4864 binds with high affinity to the rat receptor but has low affinity for the bovine PBR. Photolabeling with an isoquinoline ligand, [3H]PK 14105, identifies a 17-kDa protein, the PBR isoquinoline binding protein (PBR/IBP), in both species. To further elucidate the role of the PBR/IBP in determining PBR benzodiazepine and isoquinoline binding characteristics, the bovine PBR/IBP was cloned and expressed. Using a cDNA encoding a rat PBR/IBP to screen a fetal bovine adrenal cDNA library, a bovine cDNA encoding a polypeptide of 169 residues was cloned. The bovine and rat PBR/IBPs had similar hydropathy profiles exhibiting five potential transmembrane domains. Transfecting the cloned bovine PBR/IBP cDNA into COS-7 cells resulted in an 11-fold increase in the density of high affinity [3H]PK 11195 binding sites which had only low affinity for Ro5-4864. Expression of the bovine PBR/IBP yields a receptor which is pharmacologically distinct from both endogenous COS-7 PBR and the rat PBR based on the affinity for several N1-alkyl-1,4-benzodiazepine ligands. These results suggest the PBR/IBP is the minimal functional component required for PBR ligand binding characteristics and the different protein sequences account for the species differences in PBR benzodiazepine ligand binding.

Comparative effects of single intraperitoneal or oral doses of sodium arsenate or arsenic trioxide during in utero development.

Numerous studies have suggested that single-day intraperitoneal (IP) injection of inorganic arsenic results in failure of neural tube closure and other malformations in rats, hamsters, and mice. Most of these studies involved treatment of limited numbers of animals with maternally toxic doses of arsenic (generally As(V)), without defining a dose-response relationship. In the present Good Laboratory Practice-compliant study, sodium arsenate (As(V)) was administered IP and arsenic trioxide (As(III)) was administered either IP or orally (by gavage) on gestational day 9 to groups of 25 mated Crl:CD(R)(SD)BR rats. Only at dose levels that caused severe maternal toxicity, including lethality, did IP injection of arsenic trioxide produce neural tube and ocular defects; oral administration of higher doses of arsenic trioxide caused some maternal deaths but no treatment-related fetal malformations. In contrast, IP injection of similar amounts of sodium arsenate (based on the molar amount of arsenic) caused mild maternal toxicity but a large increase in malformations, including neural tube, eye, and jaw defects. In summary, neural tube and craniofacial defects were observed after IP injection of both As(V) and As(III); however, no increase in malformations was seen following oral administration of As(III), even at maternally lethal doses. These results demonstrate that the frequently cited association between prenatal exposure to inorganic arsenic and malformations in laboratory animals is dependent on a route of administration that is not appropriate for human risk assessment.