RNA Adducts Research Articles

Characterization of recombinant HIV-1 Tat and its interaction with TAR RNA.

Recombinant HIV-1 Tat (Tat 1-86) has been purified from the cytoplasmic fraction of Escherichia coli without the use of protein denaturants or chaotropic agents. Chloroquine-mediated uptake of the purified protein into cells resulted in transactivation of the HIV LTR promoter. Tat retains 1.64 mol of Zn2+/mol of protein by atomic absorption spectroscopy. Circular dichroism measurements indicated that the structure of recombinant Tat contains 15-20% alpha-helix. Filter binding assays showed that Tat binds to a 63-nucleotide target TAR RNA with a dissociation constant (Kd) of 10 nM at 25 degrees C, 0.05 M ionic strength, pH 7.5, in a 1:1 Tat-TAR RNA stoichiometry. Nonelectrostatic interactions provide the principal source of free energy of association. While the pH optimum occurs over a wide H+ concentration, the salt dependence of Kd indicates formation of a single ion pair. UV-induced protein-RNA cross-linking produced a labeled Tat-TAR RNA adduct, indicating that direct contact occurred between the Tat protein and TAR RNA.

Colloidal gold ultraimmunocytochemical localization of DNA and RNA adducts in rat hepatocytes.

The localization of DNA and RNA adducts was studied at the ultrastructural level using antibodies directed against O6-methylguanine (O6-metG) and the protein A-gold technique. Primary rat hepatocyte cultures were exposed for 2 h to 5 mM N-nitrosodimethylamine (NDMA). In NDMA-treated cells, the O6-metG-induced immunoreactive sites do not appear at random but seem to be concentrated in the nucleus, and in the cytoplasm, in areas rich in rough endoplasmic reticulum (RER) elements. Mitochondria were not significantly labelled. Untreated control preparations showed no specific immunogold labelling. After RNase digestion of ultrathin sections obtained from cells exposed to NDMA and subsequent immunogold labelling, most of the immunolabelling in the cytoplasm had disappeared, and that over the nucleus had only been slightly reduced, as compared to undigested specimens from NDMA-treated cultures. After similar digestion with DNase, a strong reduction of the labelling of the nucleus was observed, but labelling of the cytoplasm was practically unaffected by this enzymatic treatment, as compared to what was observed in undigested preparations of NDMA-treated hepatocytes. The results provide evidence of preferential formation of O6-metG at the DNA and RNA levels, in the nucleus and cytoplasm RER, respectively. Furthermore, this study demonstrates the applicability of the high-resolution protein A-gold technique for ultrastructural detection of nucleic acid adducts in NDMA-treated hepatocytes using affinity-purified anti-O6-metG polyclonal antibodies.

DNA adduct formation in liver following the administration of [3H]2-nitrofluorene to rats in vivo

The formation of RNA and DNA adducts by the environmental pollutant 2-nitrofluorene (2-NF) has been investigated in rat liver in vivo. The adduct pattern was studied after trifluoroacetic acid hydrolysis of DNA or RNA, followed by analysis of the adducts by HPLC. This was also done by enzymatic hydrolysis of DNA, followed by 32P-postlabeling. Both after oral and i.v. administration of [3H]2-NF, one major adduct was found. This adduct did not co-migrate with one of the known adducts of 2-(acetyl)-aminofluorene, N-deoxyguanosin-8-yl-2-aminofluorene (dG-C8-AF), which could have been formed after nitroreduction of 2-NF. 32P-Postlabeling revealed that two minor adducts were also formed, one of which was dG-C8-AF. The observation that the major adduct was also formed after i.v. administration of 2-NF to bile duct-catheterized rats makes a role for the intestinal microflora in the formation of this adduct very unlikely. In vitro experiments with inhibitors of the enzyme epoxide hydrolase indicated that epoxidation of 2-NF may play a role in the microsomal bioactivation of this compound.

Localization of DNA adducts induced by N‐acetoxy‐N‐2‐acetylaminofluorene in chinese hamster ovary cells using electron microscopy and colloidal gold

DNA adduct induction by N-acetoxy-N-2-acetylaminofluorene (N-Ac-AAF) has been investigated in Chinese hamster ovary (CHO) cells using immunoelectron microscopy. The major RNA and DNA adducts, N-(guanosin-8-yl)-2-aminofluorene (G-C8-AF) and N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF), were localized with a rabbit anti-G-C8-AF antiserum and colloidal gold cytochemistry. Appropriate controls, including incubation of untreated cells with normal rabbit serum and immunogen-absorbed serum, demonstrated that colloidal gold deposits were indicative of the presence of adducts. The localization of gold particles in close association with nuclear chromatin revealed high concentration of adducts in DNA and RNA of nuclei. Morphometric evaluation of adduct formation in organelles of from different carcinogen exposures showed that 85-88% of total adducts were concentrated in nuclei. DNA adducts remaining in nuclei after RNAse treatment appeared to concentrate in heterochromatic areas, and these areas contained 59% of bound gold particles by morphometry. A total of 137-178 particles were found in nuclei of treated cells vs. 15-26 in the surrounding cytoplasm. Treated cells incubated with normal rabbit serum or specific adduct-absorbed serum showed 19-34 particles for all cellular compartments.

Differential repair and replication of damaged DNA in ribosomal RNA genes in different CHO cell lines

We studied the repair of psoralen adducts in the pol I-transcribed ribosomal RNA (rRNA) genes of excision repair competent Chinese hamster ovary (CHO) cell lines, their UV sensitive mutant derivatives, and their UV resistant transformants, which express a human excision repair gene. In the parental cell line CHO-AA8, both monoadducts and interstrand crosslinks are removed efficiently from the rRNA genes, whereas neither adduct is removed in the UV sensitive derivative UV5; removal of both adducts is restored in the UV resistant transformant CHO-5T4 carrying the human excision repair gene ERCC-2. In contrast, removal of psoralen adducts from the rRNA genes is not detected in another parental CHO cell line CHO-9, neither in its UV sensitive derivative 43-3B, nor in its UV resistant transformant 83-G5 carrying the human excision repair gene ERCC-1. In contrast to such intergenomic heterogeneity of repair, persistence of psoralen monoadducts during replication of the rRNA genes occurs equally well in all CHO cell lines tested. From these data, we conclude that: 1) the repair efficiency of DNA damage in the rRNA genes varies between established parental CHO cell lines; 2) the repair pathways of intrastrand adducts and interstrand crosslinks in mammalian cells share, at least, one gene product, i.e., the excision repair gene ERCC-2; 3) replicational bypass of psoralen monoadducts at the CHO rRNA locus occurs similarly on both DNA strands.

The formation of covalent adducts between benzo[a]pyrenediol epoxide and RNA: structural analysis by mass spectrometry

Racemic 7-r,8-t-dihydroxy-9-t,10-t-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene was reacted with yeast RNA. Modified nucleosides were isolated and resolved by high-performance liquid chromatography; nine adduct peaks were collected for analysis. The bases in these adducts were identified by comparing their retention times with those of adducts from poly(G), poly(A), and poly(C). These samples gave two major and two minor Guo adducts, four major Ado adducts, and at least four Cyd adducts. The relative efficiencies of adduct formation with the polyribonucleotides were poly(G) greater than yeast RNA greater than poly(A) greater than poly(C). Fluorescence measurements show that emission from Guo adducts is strongly quenched relative to that from Ado adducts. Liquid secondary ion mass spectrometry (LSIMS) of underivatized samples and electron-impact mass spectrometry (EIMS) of permethyl derivatives were used to confirm the base identities and establish the alkylation sites of the RNA adducts. Unique nitrogen-containing hydrocarbon fragments that were observed with all samples by EIMS establish that in each adduct analyzed the C-10 position of the hydrocarbon is linked to the exocyclic amino group of the base. This suggested that the multiple adducts formed with each base are diastereomers derived from cis/trans epoxide ring opening of the (+) and (-) enantiomers of the carcinogen. Several adducts exhibited molecular ions by both LSIMS and EIMS. Large fragments observed by EIMS usually resulted from the loss of CH3OH, CH3O., CH2O, CH3., and H. from the molecular ion. Major fragmentation pathways also resulted in formation of nucleoside, base, ribose, hydrocarbon, and base-hydrocarbon ions. Each of these major ions in turn resulted in further characteristic fragmentation patterns.

ChemInform Abstract: Synthesis of N6‐Adenosine Adducts Expected from Cyclopenta‐Ring Activation of Acenaphthylene and Aceanthrylene.

AbstractAcenaphthenone (I) is converted to a mixture of the trans‐ and cis‐acenaphthenaminols (V) and (VI) with the predominance of (V).

Metabolic activation of 9-hydroxymethyl-10-methylanthracene and 1-hydroxymethylpyrene to electrophilic, mutagenic and tumorigenic sulfuric acid esters by rat hepatic sulfotransferase activity

Our previous studies on 7-hydroxymethyl-12-methylbenz[a]anthracene and 6-hydroxymethylbenzo[a]pyrene showed that cytosolic sulfotransferase activity plays a major role in the formation of hepatic benzylic DNA and RNA adducts by these carcinogens in rats. In the present study, we found similar sulfotransferase activity in rat liver cytosol which activates 9-hydroxymethyl-10-methylanthracene (HMA) and 1-hydroxymethylpyrene (HMP) to electrophilic sulfuric acid ester metabolites. Thus, incubation of these nonbay region hydrocarbons with calf thymus DNA in the presence of liver cytosol fortified with the sulfo-group donor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) produced benzylic DNA adducts that were chromatographically identical to those obtained by the reactions of the corresponding sulfuric acid esters with deoxyguanosine and deoxyadenosine. These adducts were also produced in the livers of infant rats injected i.p. with 0.25 mumol/g body wt of HMA or HMP. Administration of comparable doses of 9-sulfooxymethyl-10-methylanthracene (SMA) and 1-sulfooxymethylpyrene (SMP) resulted in much higher levels of hepatic benzylic DNA adducts than did the parent hydroxymethyl hydrocarbons. Both HMA and HMP induced His+ revertants in Salmonella typhimurium TA98 when preincubated with these bacteria in the presence of rat liver cytosol and PAPS. This sulfotransferase-mediated mutagenicity of HMA and HMP was reduced by dehydroepiandrosterone, an inhibitor of hepatic sulfotransferase activity for these hydrocarbons. SMA and SMP were directly mutagenic and their intrinsic bacterial mutagenicity was inhibited by glutathione (GSH) and GSH-S-transferase activity. Chloride ion at physiological concentrations enhanced the bacterial mutagenicity of SMA through the formation of 9-chloromethyl-10-methylanthracene as previously observed for SMP by Henschler et al. In contrast to the higher mutagenicity of 1-chloromethylpyrene (CMP) than SMP in bacteria, CMP formed smaller amounts of hepatic benzylic DNA adducts in rats than the sulfuric acid ester. SMA and SMP were weak skin tumor initiators in the mouse, but they were more active than HMA and HMP in this regard.

Sulfation-dependent formation of N-acetylated and deacetylated DNA adducts of N-hydroxy-4-acetylamlnobiphenyl in male rat liver in vivo and in isolated hepatocytes

Administration of 3H-labeled N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) to male Wistar rats with or without prior partial hepatectomy (PH) resulted in covalent binding of 3H activity to liver macromolecules. Pretreatment with the sulfotransferase inhibitor pentachlorophenol (PCP) 45 min before administration of the arylhydroxamic acid strongly decreased the covalent binding. Analysis of aminobiphenyl adducts after TFA hydrolysis of DNA and RNA showed that PCP decreased the formation of both the N-acetylated adduct N-[deoxy)guanosin-8-yl)-4-acetylaminobiphenyl [(d)G-C8-AABP] and the deacetylated adduct N-[deoxy)-guanosin-8-yl)-4-aminobiphenyl [(d)G-C8-ABP] by 60-80%. In incubations with hepatocytes from male Wistar or Sprague-Dawley rats, omission of inorganic sulfate also strongly decreased the covalent binding of 3H-labeled N-OH-AABP to RNA and protein. Analysis of RNA adducts showed a 70-80% decrease in the formation of G-C8-ABP in the absence of sulfate. Another, as yet unidentified, adduct was only slightly decreased. Similar results were obtained with the structurally related carcinogen N-hydroxy-4'-fluoro-4-acetylaminobiphenyl (N-OH-FAABP). Pretreatment with PCP decreased the incidence of gamma-glutamyltranspeptidase-positive foci in the liver of male rats when analyzed 30 days after a single injection of N-OH-AABP or N-OH-FAABP by 60 and 80% respectively. Thus, both N-acetylated and deacetylated RNA and DNA adducts of N-OH-AABP in rat liver are formed by sulfation and this metabolic activation pathway is responsible for the formation of genotoxic metabolites involved in the generation of preneoplastic cells.

Synthesis of N 6-adenosine adducts expected from cyclopenta-ring activation of acenaphthylene and aceanthrylene

cis and trans Acenaphthen-1-amine-2-ol and trans aceanthren-1-amine-2-ol react with 2-chloropurine-9-β- D -ribofuranose to yield easily separable diastereomeric mixtures of N 6-modified adenosine, corresponding to RNA adducts expected from the corresponding 1,2-oxides. These are the first examples of nucleoside adducts of cyclopentaPAH.

The role of sulfation in the metabolic activation of N-hydroxy-4′-fluoro-4-acetylaminobiphenyl

The role of sulfation in the metabolic activation of the liver carcinogen N-hydroxy-4'-fluoro-4-acetylaminobiphenyl (N-OH-FAABP) in male rat liver was investigated. N-OH-FAABP was a substrate for sulfotransferases in vitro and sulfation was inhibited by the sulfotransferase inhibitors pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP). The main metabolite of N-OH-FAABP excreted in bile in vivo, and in the isolated perfused liver, was identified as the N-O-glucuronide conjugate. Inhibition of sulfation in vivo by PCP or DCNP, or in vitro by omission of inorganic sulfate, resulted in a large increase in the excretion of the N-O-glucuronide conjugate. It was estimated that at least 21% of the dose was sulfated in control animals. Inhibition of sulfation in vivo by PCP or DCNP prevented the covalent binding of N-OH-FAABP to liver (and kidney) macromolecules by 70% and 20% respectively. HPLC analysis of the fluorobiphenyl DNA and RNA adducts showed that the formation of both N-acetylated and deacetylated (deoxy)-guanosine adducts was prevented. Furthermore, omission of inorganic sulfate in the isolated perfused liver prevented the formation of all fluorobiphenyl DNA adducts by 70-80%. It is concluded that two sulfotransferase-dependent pathways exist for the metabolic activation of N-OH-FAABP in male rat liver: (i) direct sulfation of the hydroxamic acid, resulting, upon decomposition of the FAABP-N-sulfate ester, in the formation of N-acetylated DNA adducts and (ii) deacetylation followed by sulfation of the hydroxylamine to FABP-N-sulfate, leading to the formation of deacetylation DNA adducts.

Hepatic DNA and RNA adduct formation from the carcinogen 7-hydroxymethyl-12-methylbenz[a]anthracene and its electrophilic sulfuric acid ester metabolite in preweanling rats and mice

DNA and RNA adducts that were chromatographically identical to those formed in vitro on reaction of 7-sulfooxymethyl-12-methyl-benz[a]anthracene with guanine and adenine nucleosides were formed in the livers of rats and mice given i.p. injections of 7-hydroxymethyl- or 7-sulfooxymethyl-12-methyl-benz[a]anthracene. Considerably higher levels of these hepatic adducts were obtained from the latter short-lived electrophilic ester than from the hydroxymethyl compound. These observations are consistent with the finding of rat liver cytosolic sulfotransferase activity for 7-hydroxymethyl-12-methylbenz[a]anthracene (Watabe et al., Science 215, 403, 1982). Formation of these hepatic adducts from 7-hydroxymethyl-12-methylbenz[a]anthracene was inhibited by prior administration to rats of dehydroepiandrosterone, an inhibitor of the sulfotransferase activity for this hydroxymethyl hydrocarbon.

Reaction of 2-acetylaminofluorene-N-sulfate with RNA and glutathione: evidence for the generation of two reactive intermediates with different reactivities towards RNA and glutathione

The sulfate ester of N-hydroxy-2-acetylaminofluorene (AAF-N-sulfate) is one of the reactive intermediates of this carcinogen. This ester breaks down spontaneously to a very reactive nitrenium ion, which reacts with nucleophilic groups in protein, DNA, RNA and glutathione (GSH). Reactions involving the nitrenium ion with several nucleophiles under various conditions were studied. The adduct formation to RNA was much higher in Tris-HCI buffer than in phosphate buffer (at pH 7.4), while adduct formation to deoxy-guanosine monomers was the same in both buffers. The presence of 150 mM KCI had the same decreasing effect in both cases. Ionic strength effects may be involved in these phenomena. GSH decreased RNA adduct formation by 20-45%, while other thiols were much more effective. On the other hand, RNA did not decrease the formation of GSH conjugates from AAF-N-sulfate. The decrease in RNA adduct formation by thiols corresponded with an increase in the formation of 2-acetylaminofluorene (AAF) from AAF-N-sulfate, while no N-hydroxy-AAF was formed. These results suggest that two independent reactive intermediates are formed from AAF-N-sulfate, with different reactivities towards RNA and glutathione. Possibly these intermediates are the 'hard' triplet state nitrenium ion and the 'soft' singlet state nitrenium ion. Cysteine, cysteamine and penicillamine were most effective in the inhibition of RNA adduct formation; the extent of inhibition correlated with the extent of AAF formation. The mechanisms involved are discussed.

The metabolic activation and nucleic acid adducts of naturally-occurring carcinogens: recent results with ethyl carbamate and the spice flavors safrole and estragole.

A small (approximately 30) but varied group of organic and inorganic compounds appear to be carcinogenic in both humans and experimental animals. A much larger number and wider variety of chemical carcinogens, primarily synthetic organic compounds, are known for experimental animals. These agents include a small (approximately 30) and varied group of metabolites of green plants and fungi. Many more of these carcinogens must exist in the living world. As with the synthetic carcinogens, the majority of these naturally occurring carcinogens are procarcinogens that require metabolic conversion into reactive electrophilic and mutagenic ultimate carcinogens. These strong electrophiles combine covalently and non-enzymatically with nucleophilic sites in DNAs, RNAs, proteins, and small molecules in target tissues. One or more of the DNA adducts appear to initiate carcinogenesis in an irreversible manner. The subsequent promotion step leading to gross tumours may be completed by further administration of carcinogen or by treatment with non-carcinogenic promoters. Roles for the RNA and protein adducts in the carcinogenic process have not been excluded. Recent data on the metabolic activation and reactivity in vivo of the naturally occurring carcinogens ethyl carbamate and certain of the alkenylbenzene spice flavours are illustrative of these principles. These agents can initiate the carcinogenic process in male mouse liver with small doses given prior to weaning. Subsequent growth of the liver and male hormonal factors appear to function as promoters leading to gross hepatic tumors after one year. Reactive electrophilic metabolites of ethyl carbamate and of safrole and estragole and their nucleic acid adducts formed during initiation in mouse liver have been characterized.

A rapid technique for estimating DNA binding, used to evaluate 1-nitropyrene adduct formation.

A simple and rapid procedure for estimating binding of radio-labelled material to DNA and protein is described. Protein was extracted from lysed rabbit alveolar macrophages with chloroform: iso-amyl-alcohol:phenol extraction. Nucleic acids were precipitated from the lysate, and hydrolysed with protease and NaOH to remove residual protein and RNA respectively. Bound radioactivity was quantitated by precipitation of DNA onto glass fiber filters. Protein labelled with 3H-leucine and DNA and RNA adducts formed from 1-nitro[14C]pyrene by xanthine oxidase were used to define this procedure. 14C was shown to be bound to endogeneous protein and DNA isolated from rabbit alveolar macrophages that had been incubated with 1-nitro[14C]pyrene.

Identification of novel 7,12-dimethylbenz[a]anthracene adducts in cellular ribonucleic acid.

The interaction of guanosine with 7,12-dimethylbenz[a]anthracene (DMBA) 5,6-oxide under alkaline conditions resulted in the formation of six derivatives. These six compounds were cochromatographed with nucleosides obtained by hydrolysis of RNA isolated from rat liver cells treated with [3H]DMBA. The cochromatography showed that three of these adducts were formed in cellular RNA. The three products constituted less than 5% of the total nucleoside-DMBA adducts as shown by chromatography on Sephadex LH-20 and high-pressure liquid chromatography. In one of them, the 2'-hydroxy group of the ribose moiety of guanosine was linked to the C-5, and in the second, to the C-6 position of the DMBA 5,6-oxide residue. In the third derivative, the C-8 position of guanosine was linked to the C-5 of the DMBA 5,6-oxide moiety. These results show, for the first time, modifications of the ribose moiety and of the guanine residue at the C-8 position in the cellular RNA by a metabolite of a polycyclic hydrocarbon.

Arylhydroxylamine-induced ribonucleic acid chain cleavage and chromatographic analysis of arylamineribonucleic acid adducts

Reaction of N-hydroxy-2-aminofluorene ( N-OH-AF) with rRNA at pH 5.0 decreased the molecular weight of the polynucleotide. Toluene-soluble aryl derivatives were released on hydrolysis of fluorenylamine- and biphenylamine-substituted RNA by treatment with venom phosphodiesterase and alkaline phosphatase. These data suggested that arylhydroxylamines, activated by incubation at pH 5.0 or by enzymatic O-acetylation, might react with the phosphate group of RNA to give unstable phosphate triesters. Spontaneous hydrolysis of these triesters would result in cleavage of the polynucleotide chain. Further enzymatic hydrolysis of the phosphate esters would yield nonpolar arylamine derivatives. Enzymatically degraded 4-aminobiphenyl(ABP)-RNA adducts were examined by high performance liquid chromatography (HPLC) for the presence of a putative phosphorylated adduct. Synthetic standards of the C-8-guanosine monophosphate-ABP adduct (ABP-GMP) and o-aminobiphenyl- O-phosphate were used as markers in the analysis of the digested RNA. A phosphate adduct of ABP was undetectable by these methods. The data also indicated that the ABP-GMP formed in the acyltransferase-mediated binding of N-hydroxy-4-acetylaminobiphenyl ( N-OH-AABP) to RNA is readily degraded during the enzymatic digestion of the RNA adduct.

The binding of N-hydroxy-2-acetylaminofluorene to DNA and repair of the adducts in primary rat hepatocyte cultures.

Primary cultures of rat hepatocytes were exposed to [ring-3H]-N-hydroxy-2-acetylaminofluorene (N-OH-AAF) for 4 h, and the RNA and DNA nucleoside adducts were isolated and identified by h.p.l.c. The DNA adducts were shown to be N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF), N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF), and 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene (dG-N2-AAF), while the RNA adducts were N-(guanosin-8-yl)-2-acetyl-aminofluorene, and N-(guanosin-8-yl)-2-aminofluorene. The removal of these adducts was measured up to 38 h following the cessation of exposure of the hepatocytes to N-OH-AAF. The dG-C8-AAF adduct was removed with a half-life of approximately 10 h, while dG-N2-AAF and dG-C8-AF remained constant for 14 h, followed by a slow rate of removal. The dG-C8-AAF adduct initially composed about 60% of the total DNA adducts of primary hepatocytes in contrast to the 20% found in liver in vivo. The formation of the 3 DNA adducts and the different rates of repair indicate that primary cultured rat hepatocytes may be a valuable system to study initiation of liver carcinogenesis by N-OH-AAF.

DNA and RNA adducts formed in hamster embryo cell cultures exposed to benzo[a]pyrene.

DNA and RNA adducts formed in hamster embryo cell cultures exposed to benzo[a]pyrene.

Structures of benzo(a)pyrene--nucleic acid adducts formed in human and bovine bronchial explants.

PUBLICATION by Sims et al. of evidence that the 7,8-dihydrodiol-9,10-oxide of benzo(a)pyrene (BP) is a metabolic intermediate in the covalent binding of this ubiquitous polycyclic aromatic hydrocarbon to DNA in hamster embryo cells1 was followed by many related publications2. Grover et al.3 also obtained evidence that the same derivative is involved in the binding of BP to the DNA of human bronchial explants, although details of the specific isomer involved and of the structure of the adduct were not reported. We describe here studies on RNA and DNA adducts formed by human bronchial explants and provide evidence that the structures of the major adducts are similar to those formed in the analogous bovine system.