Swine brucellosis: current perspectives.

Brucella suis is a significant zoonotic species that is present in domestic livestock and wildlife in many countries worldwide. Transmission from animal reservoirs is the source of human infection as human-to-human transmission is very rare. Although swine brucellosis causes economic losses in domestic livestock, preventing human infection is the primary reason for its emphasis in disease control programs. Although disease prevalence varies worldwide, in areas outside of Europe, swine brucellosis is predominantly caused by B. suis biovars 1 and 3. In Europe, swine are predominantly infected with biovar 2 which is much less pathogenic in humans. In many areas worldwide, feral or wild populations of swine are important reservoir hosts. Like other Brucella spp. in their natural host, B. suis has developed mechanisms to survive in an intracellular environment and evade immune detection. Limitations in sensitivity and specificity of current diagnostics require use at a herd level, rather for individual animals. There is currently no commercial vaccine approved for preventing brucellosis in swine. Although not feasible in all situations, whole-herd depopulation is the most effective regulatory mechanism to control swine brucellosis.

Arsenite uptake and metabolism by rat hepatocyte primary cultures in comparison with kidney- and hepatocyte-derived rat cell lines.

Biotransformation by methylation to monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) influences inorganic arsenical toxicity, which is often investigated in cultured cells. Arsenic (III) uptake and methylation was assessed in rat hepatocytes in primary culture and in three established rat cell lines (hepatoma-derived McA-RH 7777 cells and H4-II-EC-3 cells, and kidney epithelium-derived NRK-52E cells) to compare their use as model systems for arsenite metabolism. Incubation of all cell types with 0.27, 0.67, 1.33, 2.67, or 6.67 microM As(III) concentrations resulted in concentration-dependent arsenic uptake and biomethylation. Arsenic uptake by the NRK-52E cells was initially slower than that of the other cells, but by 8 h, total uptake was similar in all cell types. At the lowest arsenite concentration, the percentages of total arsenic methylated to MMA and DMA by the hepatocytes and the McA-RH 7777 cells were similar (67 and 66%); methylation by the H4-II-EC-3 cells was somewhat lower (52%), and methylation by the kidney-derived NRK-52E cells was much lower (15%). Total arsenic methylation was inhibited in the cell lines, but not in the hepatocytes, at the highest arsenite concentrations. In all cases, exposure to increased arsenite concentrations inhibited conversion of MMA to DMA much more than it affected the initial methylation step (inorganic arsenite to MMA). These results indicate that rat hepatocytes in primary culture and established rat hepatoma-derived cell lines are similar in their abilities to accumulate and methylate arsenic to MMA and DMA at environmentally relevant arsenic concentrations in the medium. They differed from the kidney epithelium-derived cells, which exhibited substantially lower biomethylation activity.

Construction of an isogenic leukotoxin deletion mutant of Pasteurella haemolytica serotype 1: characterization and virulence.

Allelic replacement was used to generate two isogenic lktA deletion mutants of Pasteurella haemolytica serotype 1 that were incapable of synthesizing leukotoxin (Lkt). Southern blot data confirmed that lktA sequences were absent in the two P. haemolytica deletion mutants. Culture supernatants and whole cell lysates from the wild type P. haemolytica, D153 parent strain, but not the lktA deletion mutants, contained immunoreactive and bioactive leukotoxic protein. In addition, only the parent strain was haemolytic when grown on bovine and sheep blood agar plates. Virulence of the lktA deletion mutant, lktA 77, was compared with the parent in an experimentally infected calf model of pneumonic pasteurellosis. Results revealed significant reduction in virulence in the lktA mutant as measured by clinical and lung lesion scores. Notable differences in histological changes such as markedly reduced necrosis and lack of leukocyte degeneration occurred in calves infected with the lktA mutant in comparison with those infected with the parent wild-type strain. Thus, it appears that leukotoxin plays a important role in the pathogenesis of lung injury in bovine pneumonic pasteurellosis.

Morphometric and histopathologic analysis of lymphoid depletion in murine spleens following infection with Brucella abortus strains 2308 or RB51 or an htrA deletion mutant.

BALB/C mice were inoculated intraperitoneally with suspensions of Brucella abortus strains 2308 or RB51 or an htrA mutant. Spleens were examined on postinoculation day (PID) 2, 4, 7, 10, 15, 21, 30, and 60. Brucellae were cultured in high numbers from the spleens of mice infected with strains 2308 or htrA through PID 60; however, mice infected with strain RB51 cleared the infection between PID 30 and PID 60. Histopathologic changes in spleens from 2308-infected mice were characterized by marked accumulations of macrophages, which expanded marginal zones beginning as early as PID 7 and persisting through PID 60. Morphometric analysis showed a decrease in splenic white pulp in 2308-infected mice at PID 10, which correlated with the peak of bacterial infection. Although this decrease was significant (P < 0.05) when compared with values at the previous (PID 7) and the following (PID 15) time periods, it was not significantly different from white pulp values noted at PID 2 or PID 4 or the values for control spleens. Spleens from RB51-infected mice showed only mild to moderate accumulations of macrophages in marginal zone areas during the peak of RB51 infection (PID 7-10). Morphometric analysis of RB51-infected spleens showed a decrease in white pulp area, which coincided with peak bacterial numbers. However, this decrease was not significant (P > 0.05). Spleens from mice infected with the htrA mutant showed moderate to marked accumulations of macrophages in marginal zone areas, which persisted through PID 60. Multifocal necrosis in lymphoid follicles as early as PID 4 was seen in both htrA and 2308 infection. Morphometric analysis of htrA-infected spleens revealed no significant decrease in white pulp and no obvious correlation with bacterial numbers in the spleen. These results suggest that virulent B. abortus does not induce lymphoid depletion significantly below those values seen in noninfected mice; thus, the possible role of lymphoid depletion in the pathogenesis of brucellosis remains questionable.

Domain structure and conformation of histidine-proline-rich glycoprotein.

The complete primary structure of rabbit plasma histidine-proline-rich glycoprotein (HPRG), also known as histidine-rich glycoprotein, was determined by a combination of cDNA and peptide sequencing. Limited proteolysis with plasmin yielded three disulfide-linked fragments that were further purified. Reduction of the disulfide bonds with dithiothreitol under nondenaturing conditions releases the central, histidine-proline-rich domain, which contains 15 tandem repeats of the pentapeptide [H/P]-[H/P]PHG. The N-terminal fragment (295 amino acids), consisting of two cystatin-like modules, is bound to the proline-rich C-terminal fragment (105 amino acids) via a buried disulfide bond whose reduction requires prior denaturation. Far-UV circular dichroism spectra revealed beta-sheet with some alpha-helix, polyproline-II helix, and random coil in the secondary structure of the N-terminal, central, and C-terminal domains, respectively. The modular architecture of HPRG suggests that it may have several independent binding sites and that its biological role may be to bring two or more ligands together. The histidine-proline-rich domain, which contains 34 of the 53 histidine residues of HPRG, binds heparin and has an isoelectric point of 7.15 and a relatively high apparent pKa (7.0) of its histidine residues, and thus it probably mediates the interaction between HPRG and heparin, which is strikingly sensitive to pH in the range 7.0-7.4 [Peterson et al. (1987) J. Biol. Chem. 262, 7567-7574]. Solvent perturbation and second-derivative UV spectroscopy of HPRG revealed changes in the environment of tryptophan residues upon lowering the pH. This transition had a midpoint at pH 6.0 and required the disulfide bond bridging the histidine-proline-rich domain to the N/C fragment. The data are consistent with the mutual repulsion of protonated histidine residues in the histidine-proline-rich region causing a conformational change transmitted to the rest of the molecule via the disulfide bond.

Cloning, characterization and construction of htrA and htrA-like mutants of Brucella abortus and their survival in BALB/c mice.

A genomic library of Brucella abortus S2308 was screened for expression of recombinant proteins recognized by sera from mice and from cattle infected with B. abortus. A positive clone, BA1, expressing a 50 kDa peptide was recognized by both sera. Plasmid pBA1, isolated from BA1, was shown by restriction enzyme digestion to possess a 1.9 kb insert. The nucleotide sequence of the pBA1 insert revealed an open reading frame with of 1539 bases with a coding capacity of 513 amino acids and a predicted molecular weight of 50,992. The predicted amino acid sequence showed 37% identity to E. coli HtrA, a temperature inducible serine protease. A second B. abortus htrA gene, designated htrA-like, was identified on a different cloned fragment that also encoded B. abortus recA. The nucleotide sequence of the htrA-like gene revealed an open reading frame of 1422 nucleotides with a coding capacity of 474 amino acids and a predicted molecular weight of 50,155. The deduced amino acid sequence of the htrA-like gene showed 42% and 36% identity with B. abortus and E. coli HtrAs respectively. Western blotting of E. coli lysate containing the htrA-like gene was not recognized by sera from B. abortus-infected cattle or mice. B. abortus htrA but not htrA-like relieved the temperature sensitive phenotype and permitted growth of an E. coli htrA mutant at 42 degrees C. B. abortus htrA and htrA-like mutants were constructed and their survival and growth in BALB/c mice was compared to the parental strain S2308.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a restriction endonuclease, PhaI, from Pasteurella haemolytica serotype A1 and protection of heterologous DNA by a cloned PhaI methyltransferase gene.

Pasteurella haemolytica is the leading cause of economic loss to the beef cattle industry in the United States and an important etiologic agent worldwide. Study of P. haemolytica is hindered by researchers' inability to genetically manipulate the organism. A new restriction endonuclease, PhaI, an isoschizomer of SfaNI (R. J. Roberts, Methods Enzymol. 65:19-36, 1980), was isolated from P. haemolytica serotype 1, strain NADC-D60, obtained from pneumonic bovine lung. PhaI recognizes the 5-base nonpalindromic sequences 5'-GCATC-3' and 5'-GATGC-3'. Cleavage occurs 5 bases 3' from the former recognition site and 9 bases 5' from the latter recognition site. A gene encoding a methyltransferase which protects against PhaI cleavage was cloned from P. haemolytica NADC-D60 into Escherichia coli. Whereas unmethylated plasmid DNA containing a P. haemolytica origin of replication was unable to transform P. haemolytica when introduced by electroporation, the same plasmid DNA obtained from E. coli which contained a cloned PhaI methyltransferase gene could do so. The data indicate that PhaI is an effective barrier to the introduction and establishment of exogenous DNA in P. haemolytica.

Molecular gene cloning and nucleotide sequencing and construction of an aroA mutant of Pasteurella haemolytica serotype A1.

The aroA gene of Pasteurella haemolytica serotype A1 was cloned by complementation of the aroA mutation in Escherichia coli K-12 strain AB2829. The nucleotide sequence of a 2.2-kb fragment encoding aroA predicted an open reading frame product 434 amino acids long that shows homology to other bacterial AroA proteins. Several strategies to inactivate aroA were unsuccessful. Gene replacement was finally achieved by constructing a replacement plasmid with aroA inactivated by insertion of a P. haemolytica ampicillin resistance fragment into a unique NdeI site in aroA. A hybrid plasmid was constructed by joining the aroA replacement plasmid with a 4.2-kb P. haemolytica plasmid which encodes streptomycin resistance. Following PhaI methylation, the replacement plasmid was introduced by electroporation into P. haemolytica NADC-D60, a plasmidless strain of serotype 1A. Allelic exchange between the replacement plasmid and the chromosome of P. haemolytica gave rise to an ampicillin-resistant mutant which grew on chemically defined P. haemolytica medium supplemented with aromatic amino acids but failed to grow on the same medium lacking tryptophan. Southern blot analysis confirmed that aroA of the mutant was inactivated and that the mutant was without a plasmid.

Sequence and characterization of an insertion sequence, IS711, from Brucella ovis.

The nucleotide (nt) sequence of a previously discovered insertion in Brucella ovis was determined and found to have the hallmarks of an insertion sequence (IS). The element, designated IS711, of 842 bp, is similar in G + C content to that of the Brucella genome and is bounded by 20-bp imperfect inverted repeats (IR). The element appears to duplicate the nt TA of a consensus target site, YTAR (R, purines; Y, pyrimidines). When the complete nt sequence of four elements and 300 bp of the 3' ends of five other elements were compared to IS711 and to each other, minor nt sequence variations were found amongst most of them. Similar to several other transposable elements, IS711 has overlapping ORFs rather than one long ORF extending the length of the element. Even though only ten B. ovis IS711 elements were characterized, in three cases we found these elements flanked by either identical or similar nt sequences. This suggests that some target sites are hot spots for insertion and that some of the elements may be duplicated by mechanisms other than transposition. No DNA or protein database entries had an obvious resemblance to either IS711 or its deduced gene products.

Immune responses and protection against infection and abortion in cattle experimentally vaccinated with mutant strains of Brucella abortus.

Twenty-four 10-month-old Polled Hereford heifers were inoculated SC with live cells of one of the following strains of Brucella abortus: S19 delta 31K (n = 4), S19 delta SOD (n = 4), RB51 (n = 4), and strain 19 (n = 6); controls (n = 6) were given saline solution. Heifers given the deletion mutants S19 delta 31K and S19 delta SOD, and those given strain 19 developed antibody responses to B abortus and cutaneous reactions to brucellin. Heifers given strain RB51 did not develop antibodies that reacted in the standard tube agglutination test, but sera reacted in tests, using an antibody dot-blot assay containing RB51 antigen. The S19 delta 31K and S19 delta SOD strains of B abortus isolated from lymph node tissue after vaccination did not differ genetically from the master stock strain. All heifers were bred naturally at 16 to 17 months of age, and were challenge-exposed intraconjunctivally with virulent B abortus strain 2308 during the fifth month of pregnancy. All vaccinated heifers were protected (ie, none aborted and none had B abortus isolated from their tissues after parturition). Calves born from vaccinated dams were free of B abortus. Antibody responses in heifers after challenge exposure were an indicator of immunity. All 5 control heifers (nonvaccinated) developed serum antibodies after challenge exposure; 3 aborted, and 1 delivered a small, weak calf at 8.5 months of gestation. Thus live mutant strains of B abortus can induce protective immunity when given at 10 months of age, and strain RB51 is a strong candidate for further testing.

Construction of a Brucella abortus RecA mutant and its survival in mice.

To determine if RecA plays a role in the virulence of Brucella abortus, a B. abortus RecA mutant was constructed and its survival was examined in mice. The recA gene was cloned from a B. abortus genomic DNA library by complementation of an Escherichia coli recA mutant in the presence of methyl methanesulfonate (MMS). The nucleotide sequence of recA was determined and the deduced protein sequence possesses extensive conservation with other RecA proteins of Gram-negative bacteria. A deletion plasmid was constructed in a suicide vector by deleting a segment of recA and inserting a kanamycin resistance gene. The deletion plasmid was introduced into B. abortus strain 2308, a virulent strain, by electroporation. Replacement of recA with the kanamycin resistance fragment was confirmed by Southern blot analysis. The RecA mutant was more sensitive than the parental strain to killing by MMS. When administered intraperitoneally to BALB/c mice, numbers of bacteria per spleen were consistently lower in animals infected with the RecA mutant than with the parental strain. However, both the RecA mutant and parental strain persisted in mice through 100 days post-infection. These results indicate that RecA is not crucial for persistence of B. abortus in mice.

Bacterial survival, lymph node changes, and immunologic responses of cattle vaccinated with standard and mutant strains of Brucella abortus.

Forty-eight cattle were used in 4 experiments; 6-week-old calves in experiments 1-3 (n = 24) and 10-month-old heifers in experiment 4 (n = 24). In experiments 1-3, 7 groups of 3 calves each were inoculated SC with 5 strains of Brucella abortus: virulent strain 2308 (2 groups), vaccine strain 19 (2 groups), and mutant strains RB51. 19 delta 31K, and 19 delta SOD. Sera and lymph node tissues were examined at 2-week intervals for evidence of infection. At postinoculation (PI) week 12, 2 calves in each group were given dexamethasone for 5 days. Calves were then euthanatized and lymphoid tissue, spleen, liver, and bone marrow were examined for evidence of B abortus. Calves given strain 2308 had large numbers of bacteria in their lymph nodes, marked granulomatous lymphadenitis in the deep cortex, and loss of lymphoid cells in superficial cortical areas. In addition, they had high serum antibody titers at PI week 16. Calves given strain 19, or genetic mutants derived from strain 19, cleared bacteria from lymph nodes more rapidly, had less lymphoid destruction, and developed antibody titers that did not persist for 16 weeks. The RB51 strain (rough) was cleared most rapidly from lymphoid tissues and induced serum antibody responses only to the core of the lipopolysaccharide molecule. Treatment of calves with dexamethasone did not cause B abortus to reappear in tissues of any calves, nor did serum antibody titers increase.(ABSTRACT TRUNCATED AT 250 WORDS)

Construction of Cu-Zn superoxide dismutase deletion mutants of Brucella abortus: analysis of survival in vitro in epithelial and phagocytic cells and in vivo in mice.

Cu-Zn superoxide dismutase (SOD) deletion mutants of Brucella abortus S2308, a virulent strain, and S19, a vaccine strain, were generated by gene replacement. A deletion plasmid, pBA delta sodknr, was constructed by excising the Cu-Zn SOD gene (Cu-Zn sod) from a 2.3-kb B. abortus DNA fragment of plasmid pBA20-1527 and inserting a 1.4-kb DNA fragment encoding kanamycin resistance into the Cu-Zn sod excision site. The deletion plasmid was introduced into B. abortus by electroporation, and Southern blot analysis confirmed that the antibiotic resistance fragment had replaced Cu-Zn sod in kanamycin-resistant colonies. The survival and growth of Cu-Zn SOD mutant strains were compared with that of the parental strains in HeLa cells and in the mouse macrophagelike cell line J774. The survival and growth of the Cu-Zn SOD mutant strains were similar to those of their respective parental strains in HeLa and J774 cell lines. The kinetics of infection with these strains were examined in BALB/c mice. The splenic levels of the S19 Cu-Zn SOD mutant recovered from intraperitoneally infected BALB/c mice were approximately 10-fold lower than those of the parental strain through 26 days postinfection. Thereafter, infection sharply declined in both groups, and by 105 days postinfection, no organisms were detected. The splenic levels of the S2308 Cu-Zn SOD mutant were lower than those of wild-type S2308-infected mice. The spleen weights of mice infected with the S2308 Cu-Zn SOD mutant were consistently lower than those of wild-type S2308-infected mice. These results suggest that the antioxidant enzyme Cu-Zn SOD plays a role in the survival and pathogenicity of B. abortus in vivo.

Deletion of the BCSP31 gene of Brucella abortus by replacement.

The 31-kDa salt-extractable immunogenic protein, BCSP31, was deleted from several Brucella abortus strains by replacement with a marker gene encoding resistance to the antibiotics kanamycin and neomycin. The BCSP31 gene replacement plasmids, constructed with ColE1-derived vectors, were introduced by electroporation into B. abortus strain 19 (S19), into a rough variant of B. abortus S19, and into B. abortus S2308, and antibiotic-resistant transformants were isolated. B. abortus S19 is an attenuated strain used as a vaccine for prevention of bovine brucellosis in the United States, and B. abortus S2308 is a commonly used challenge strain. The antibiotic-resistant isolates were all obtained by recombination; none were spontaneous mutants. Loss of the gene encoding BCSP31 and presence of the marker gene were confirmed by Southern analysis. Vector sequences were either absent or linked to the genome, indicating that ColE1-derived plasmids are not maintained in B. abortus. Survival of B. abortus mutant strains in the macrophagelike cell line J774 and in HeLa cells was examined and shown to be indistinguishable from that of the parental strain.

Use of hemopexin domains and monoclonal antibodies to hemopexin to probe the molecular determinants of hemopexin-mediated heme transport.

Plasmin cleaves rabbit serum apohemopexin (Mr = 60,000) at a single site producing a heme-binding domain (I, Mr = 35,000) and a second domain (II, Mr = 25,000) (W. T. Morgan and A. Smith (1984) J. Biol. Chem. 259, 12001-12005). The absorbance spectra of heme-domain I are indicative of a bis-histidyl coordination complex with the central heme iron atom. Chemical modification of the 5 histidine residues of apo-domain I with diethylpyrocarbonate abolished heme binding, supporting this assignment. Upon binding heme, domain I migrates more rapidly in sucrose gradients, and, in sedimentation velocity experiments, the s value of domain I increases from 3.17 +/- 0.04 to 3.71 +/- 0.09, a notably large increase which indicates that the domain becomes much more compact. This conformational change which plays a pivotal role in hemopexin function requires the bis-histidyl coordination with heme iron and leads to a tighter association between domain I and domain II shown by the co-migration of heme-domain I and domain II in sucrose gradients. In turn, the association of heme-domain I with domain II increases the thermal stability of the heme-domain I chromophore. Results of binding studies using mouse hepatoma cells and isolated domains indicate that domain I not only binds heme but also plays a vital part in the hemopexin-receptor interaction. The change in conformation of domain I upon heme binding and the association between domains I and II induced by heme are both notable determinants of the strength of the hemopexin-receptor interaction, but an intact "hinge region" between the domains is not necessary for receptor binding. The importance of both domains in bringing about the transport function of hemopexin is confirmed by the ability of three (two specific for domain I and one for domain II) of seven monoclonal antibodies raised against hemopexin to inhibit the hemopexin-receptor interaction.

Interaction of hemopexin with Sn-protoporphyrin IX, an inhibitor of heme oxygenase. Role for hemopexin in hepatic uptake of Sn-protoporphyrin IX and induction of mRNA for heme oxygenase.

Sn-protoporphyrin IX (SnPP), an inhibitor of heme oxygenase and a potential therapeutic agent for neonatal hyperbilirubinemia, is bound tightly by hemopexin. The apparent dissociation constant (Kd) at pH 7.4 is 0.25 +/- 0.15 microM, but estimation of the Kd for the SnPP-hemopexin complex is hampered by the fact that at physiological pH SnPP exists as monomers and dimers, both of which are bound by hemopexin. SnPP is readily displaced from hemopexin by heme (Kd less than 1 pM). The hemopexin-SnPP interaction, like that of heme-hemopexin, is dependent on the histidine residues of hemopexin. However, as expected from the differences in the coordination chemistries of tin and iron, the stability of the histidyl-metalloporphyrin complex is lower for SnPP-hemopexin than for mesoheme-hemopexin. Nevertheless, when SnPP binds to hemopexin, certain of the ligand-induced changes in the conformation of hemopexin which increase the affinity of the protein for its receptor are produced. Binding of SnPP produces the conformational change in hemopexin which protects the hinge region of hemopexin from proteolysis, but SnPP does not produce the characteristic increase in the ellipticity of hemopexin at 231 nm that heme does. Competition experiments confirmed that human serum albumin (apparent Kd = 4 +/- 2 microM) has a significantly lower affinity for SnPP than does hemopexin. Appreciable amounts of SnPP (up to 35% in adults and 20% in neonates) would be bound by hemopexin in the circulation, and the remainder of SnPP would be associated with albumin due to the latter's high concentration in serum. Essentially no non-protein-bound SnPP is present. Importantly, SnPP-hemopexin binds to the hemopexin receptor on mouse hepatoma cells with an affinity comparable to that of heme-hemopexin and treatment of the hepatoma cells with SnPP-hemopexin causes a rapid increase in the steady state level of heme oxygenase messenger RNA. These results show that hemopexin participates in the transport of SnPP to heme oxygenase and in its regulation by SnPP.

Importance of ligand-induced conformational changes in hemopexin for receptor-mediated heme transport.

Hemopexin alters conformation upon binding heme as shown by circular dichroism (CD), but hemopexin binds the heme analog, iron-meso-tetra-(4-sulfonatophenyl)-porphine (FeTPPS), without undergoing concomitant changes in its CD spectrum. Moreover, FeTPPS, unlike heme, does not increase the compactness of the heme-binding domain (I) of hemopexin shown by an increased sedimentation rate in sucrose gradients. On the other hand, like heme, FeTPPS forms a bishistidyl coordination complex with hemopexin and upon binding protects hemopexin from cleavage by plasmin. Competitive inhibition and saturation studies demonstrate that FeTPPS-hemopexin binds to the hemopexin receptor on mouse hepatoma cells but with a lower affinity (Kd 125 nM) more characteristic of apo-hemopexin than heme-hemopexin (Kd 65 nM). This provides evidence that conformational changes produced in hemopexin upon binding heme, but not upon binding FeTPPS, are important for increasing the affinity of hemopexin for its receptor. The amount of cell-associated radiolabel from 55FeTPPS-hemopexin increases linearly for up to 90 min but at a rate only about a third of that of the mesoheme-complex. As expected from the recycling of hemopexin, more iron-tetrapyrrole than protein is associated with the Hepa cells, but the ratio of 55Fe-ligand to 125I-hemopexin is only 2:1 for FeTPPS-hemopexin compared to 4:1 for mesoheme complexes. [55Fe]Mesoheme was associated at 5 min with lower density fractions containing plasma membranes and at 30 min with fractions containing higher density intracellular compartments. In contrast, 55FeTPPS was found associated with plasma membrane fractions at both times and was not transported into the cell. Although FeTPPS-hemopexin binds to the receptor, subsequent events of heme transport are impaired. The results indicate that upon binding heme at least three types of conformational changes occur in hemopexin which have important roles in receptor recognition and that the nature of the ligand influences subsequent heme transport.

Uptake, distribution, and metabolism of trivalent arsenic in the pregnant mouse.

To investigate the distribution of trivalent arsenic (arsenite) in the pregnant rodent, CD-1 mice were dosed with sodium arsenite by ip injection or by gavage on gestation d 18 (copulation plug day = d 1). Doses were 8 (ip) or 25 (po) mg/kg, and samples of maternal blood, liver, and kidneys, as well as fetuses and pooled placentas, were analyzed for total arsenic at intervals of up to 24 h. Fetal tissue was also analyzed for relative proportions of inorganic arsenic and methylated metabolites. Arsenic uptake was significantly greater in the injected mice and their fetuses (as a proportion of the administered dose), with levels highest at 10 min to 4 h in maternal tissues and 24 h in fetuses. Peak maternal arsenic levels (as microgram/g or microgram/ml) ranged from 2.36 (blood) to 26.15 (liver) for the ip injected and 1.25 (blood) to 17.64 (liver) for the gavaged treatment group. The rate of arsenic elimination from maternal samples was not significantly influenced by administration route, with first-order elimination rate constants (k) of 0.215 and 0.234 h-1 for the po and ip dosed mice, respectively. Fetal tissue arsenic peaks were 2.10 and 0.77 micrograms/g for the ip and po treatment groups, respectively. The proportion of methylated arsenic in fetuses increased to 79% in the ip treatment group and 88% in the po group by 24 h. Such results show that much of the arsenic reaching the mouse fetus has been methylated to less toxic metabolites. They also confirm that assumptions made regarding hazard to the fetus must reflect the likelihood that a portion of any maternal dose of inorganic arsenite reaching a fetus may have been methylated, and they support previous findings that arsenite is toxic to the conceptus at lower doses when given by injection than by gavage.

Distribution, metabolism, and fetal uptake of pentavalent arsenic in pregnant mice following oral or intraperitoneal administration.

Pregnant CD-1 mice were treated with 20 (i.p.) or 40 (p.o.) mg/kg sodium arsenate on gestation day 18 (plug = day 1). Individual fetuses, pooled placentas and maternal blood, urine, liver, and kidneys were obtained from three or more litters at intervals up to 24 hours following treatment. Acid-digested samples were analyzed for total arsenic by hydride generation atomic absorption spectrophotometry. The rate of arsenic elimination from maternal samples was not influenced by administration route. First-order elimination followed a brief period of distribution, and the biological half-life was approximately 10 hours. Arsenic was found in most samples, with mean peak concentrations expressed as micrograms As/gm (wet wt.) or /ml (values listed are post-treatment sampling times in minutes or hours and concentrations for i.p. and for p.o. treated groups, respectively) as follows: fetuses-2, 3.5; 6, 0.8, placentas-2, 9.3; 1, 2.3, blood-10 minutes, 6.9; 1, 2.0, urine-1, 712; 2, 342, kidney-20 minutes, 25.4; 1, 11.0, liver-0.5, 7.9; 1, 11.7. By 24 hours, arsenic levels in fetuses and placentas had declined to 0.22 microgram/gm and 0.74 microgram/gm for i.p. and 0.33 microgram/gm and 0.57 microgram/gm for p.o. treatments, respectively. Fetal arsenic uptake and loss were more rapid following i.p. than p.o. treatments, and although the i.p. dose was only half that used p.o., peak fetal As+5 was almost fivefold higher following i.p. treatment. These results agree with the finding that oral dosing of pregnant mice with arsenate has less effect on the conceptus than does treatment by injection.(ABSTRACT TRUNCATED AT 250 WORDS)