Whole-exome sequencing identifies homozygous AFG3L2 mutations in a spastic ataxia-neuropathy syndrome linked to mitochondrial m-AAA proteases

We report an early onset spastic ataxia-neuropathy syndrome in two brothers of a consanguineous family characterized clinically by lower extremity spasticity, peripheral neuropathy, ptosis, oculomotor apraxia, dystonia, cerebellar atrophy, and progressive myoclonic epilepsy. Whole-exome sequencing identified a homozygous missense mutation (c.1847G>A; p.Y616C) in AFG3L2, encoding a subunit of an m-AAA protease. m-AAA proteases reside in the mitochondrial inner membrane and are responsible for removal of damaged or misfolded proteins and proteolytic activation of essential mitochondrial proteins. AFG3L2 forms either a homo-oligomeric isoenzyme or a hetero-oligomeric complex with paraplegin, a homologous protein mutated in hereditary spastic paraplegia type 7 (SPG7). Heterozygous loss-of-function mutations in AFG3L2 cause autosomal-dominant spinocerebellar ataxia type 28 (SCA28), a disorder whose phenotype is strikingly different from that of our patients. As defined in yeast complementation assays, the AFG3L2(Y616C) gene product is a hypomorphic variant that exhibited oligomerization defects in yeast as well as in patient fibroblasts. Specifically, the formation of AFG3L2(Y616C) complexes was impaired, both with itself and to a greater extent with paraplegin. This produced an early-onset clinical syndrome that combines the severe phenotypes of SPG7 and SCA28, in additional to other "mitochondrial" features such as oculomotor apraxia, extrapyramidal dysfunction, and myoclonic epilepsy. These findings expand the phenotype associated with AFG3L2 mutations and suggest that AFG3L2-related disease should be considered in the differential diagnosis of spastic ataxias.     

Loss of the m‐AAA protease subunit AFG3L2 causes mitochondrial transport defects and tau hyperphosphorylation

The m‐AAA protease subunit AFG3L2 is involved in degradation and processing of substrates in the inner mitochondrial membrane. Mutations in AFG3L2 are associated with spinocerebellar ataxia SCA28 in humans and impair axonal development and neuronal survival in mice. The loss of AFG3L2 causes fragmentation of the mitochondrial network. However, the pathogenic mechanism of neurodegeneration in the absence of AFG3L2 is still unclear. Here, we show that depletion of AFG3L2 leads to a specific defect of anterograde transport of mitochondria in murine cortical neurons. We observe similar transport deficiencies upon loss of AFG3L2 in OMA1‐deficient neurons, indicating that they are not caused by OMA1‐mediated degradation of the dynamin‐like GTPase OPA1 and inhibition of mitochondrial fusion. Treatment of neurons with antioxidants, such as N‐acetylcysteine or vitamin E, or decreasing tau levels in axons restored mitochondrial transport in AFG3L2‐depleted neurons. Consistently, tau hyperphosphorylation and activation of ERK kinases are detected in mouse neurons postnatally deleted for Afg3l2. We propose that reactive oxygen species signaling leads to cytoskeletal modifications that impair mitochondrial transport in neurons lacking AFG3L2.     

Nonsense mutations in the COX1 subunit impair the stability of respiratory chain complexes rather than their assembly

Respiratory chain (RC) complexes are organized into supercomplexes forming 'respirasomes'. The mechanism underlying the interdependence of individual complexes is still unclear. Here, we show in human patient cells that the presence of a truncated COX1 subunit leads to destabilization of complex IV (CIV) and other RC complexes. Surprisingly, the truncated COX1 protein is integrated into subcomplexes, the holocomplex and even into supercomplexes, which however are all unstable. Depletion of the m-AAA protease AFG3L2 increases stability of the truncated COX1 and other mitochondrially encoded proteins, whereas overexpression of wild-type AFG3L2 decreases their stability. Both full-length and truncated COX1 proteins physically interact with AFG3L2. Expression of a dominant negative AFG3L2 variant also promotes stabilization of CIV proteins as well as the assembled complex and rescues the severe phenotype in heteroplasmic cells. Our data indicate that the mechanism underlying pathogenesis in these patients is the rapid clearance of unstable respiratory complexes by quality control pathways, rather than their impaired assembly.     

Loss of m-AAA protease in mitochondria causes complex I deficiency and increased sensitivity to oxidative stress in hereditary spastic paraplegia

Mmutations in paraplegin, a putative mitochondrial metallopeptidase of the AAA family, cause an autosomal recessive form of hereditary spastic paraplegia (HSP). Here, we analyze the function of paraplegin at the cellular level and characterize the phenotypic defects of HSP patients' cells lacking this protein. We demonstrate that paraplegin coassembles with a homologous protein, AFG3L2, in the mitochondrial inner membrane. These two proteins form a high molecular mass complex, which we show to be aberrant in HSP fibroblasts. The loss of this complex causes a reduced complex I activity in mitochondria and an increased sensitivity to oxidant stress, which can both be rescued by exogenous expression of wild-type paraplegin. Furthermore, complementation studies in yeast demonstrate functional conservation of the human paraplegin-AFG3L2 complex with the yeast m-AAA protease and assign proteolytic activity to this structure. These results shed new light on the molecular pathogenesis of HSP and functionally link AFG3L2 to this neurodegenerative disease.     

Regulation of OPA1 processing and mitochondrial fusion by m-AAA protease isoenzymes and OMA1

Mitochondrial fusion depends on the dynamin-like guanosine triphosphatase OPA1, whose activity is controlled by proteolytic cleavage. Dysfunction of mitochondria induces OPA1 processing and results in mitochondrial fragmentation, allowing the selective removal of damaged mitochondria. In this study, we demonstrate that two classes of metallopeptidases regulate OPA1 cleavage in the mitochondrial inner membrane: isoenzymes of the adenosine triphosphate (ATP)–dependent matrix AAA (ATPase associated with diverse cellular activities [m-AAA]) protease, variable assemblies of the conserved subunits paraplegin, AFG3L1 and -2, and the ATP-independent peptidase OMA1. Functionally redundant isoenzymes of the m-AAA protease ensure the balanced accumulation of long and short isoforms of OPA1 required for mitochondrial fusion. The loss of AFG3L2 in mouse tissues, down-regulation of AFG3L1 and -2 in mouse embryonic fibroblasts, or the expression of a dominant-negative AFG3L2 variant in human cells decreases the stability of long OPA1 isoforms and induces OPA1 processing by OMA1. Moreover, cleavage by OMA1 causes the accumulation of short OPA1 variants if mitochondrial DNA is depleted or mitochondrial activities are impaired. Our findings link distinct peptidases to constitutive and induced OPA1 processing and shed new light on the pathogenesis of neurodegenerative disorders associated with mutations in m-AAA protease subunits.     

Molecular and functional analyses of the human and mouse genes encoding AFG3L1, a mitochondrial metalloprotease homologous to the human spastic paraplegia protein

The identification of SPG7 as the gene defective in a recessive form of spastic paraplegia has drawn attention to the yeast protein family of ATP-dependent zinc metalloproteases. The protein encoded by SPG7, paraplegin, shows high homology to members of this protein family. Recently, many mammalian ATP-dependent zinc metalloproteases have been identified and considered as possible candidates for defects in other forms of hereditary spastic paraplegia and possibly other neurodegenerative disorders. So far only a partial sequence has been available for one of those genes, ATPase family gene-3, yeast-like-1 (AFG3L1). We have carried out detailed molecular analysis of this gene and identified and characterized its mouse orthologue, Afg3l1. Our data indicate that AFG3L1 is transcribed into four mRNA isoforms that are not translated in humans. Afg3l1 encodes a protein with high homology to paraplegin and the other members of the ATP-dependent zinc metalloprotease family. Like the other ATP-dependent zinc metalloproteases, Afg3l1 localizes to the mitochondria.     

Oxidation induces ClC-3-dependent anion channels in human leukaemia cells

To test for redox regulation of anion channels in erythroid cells, we exposed K562 cells to oxidants and measured changes in transmembrane Cl(-) currents using patch-clamp, and in intracellular Cl(-) content using the Cl(-) selective dye MQAE. Oxidation with tert-butylhydroperoxide or H(2)O(2) produced a plasma membrane anion permeability with a permselectivity of NO(3)(-)>lactate(-)>gluconate(-). The permeability increase was paralleled by insertion of ClC-3 protein into the plasma membrane as evident from immunofluorescence microscopy and surface biotinylation. Down-regulation of ClC-3 protein by RNA interference as assessed by immunoblotting decreased the oxidation-stimulated permeability. In conclusion, oxidation induces surface expression of ClC-3 and activation of a ClC-3-dependent anion permeability in K562 cells.     

Regulation of mitochondrial proteostasis by the proton gradient

Mitochondria adapt to different energetic demands reshaping their proteome. Mitochondrial proteases are emerging as key regulators of these adaptive processes. Here, we use a multiproteomic approach to demonstrate the regulation of the m‐AAA protease AFG3L2 by the mitochondrial proton gradient, coupling mitochondrial protein turnover to the energetic status of mitochondria. We identify TMBIM5 (previously also known as GHITM or MICS1) as a Ca²⁺/H⁺ exchanger in the mitochondrial inner membrane, which binds to and inhibits the m‐AAA protease. TMBIM5 ensures cell survival and respiration, allowing Ca²⁺ efflux from mitochondria and limiting mitochondrial hyperpolarization. Persistent hyperpolarization, however, triggers degradation of TMBIM5 and activation of the m‐AAA protease. The m‐AAA protease broadly remodels the mitochondrial proteome and mediates the proteolytic breakdown of respiratory complex I to confine ROS production and oxidative damage in hyperpolarized mitochondria. TMBIM5 thus integrates mitochondrial Ca²⁺ signaling and the energetic status of mitochondria with protein turnover rates to reshape the mitochondrial proteome and adjust the cellular metabolism.     

Conversion of 11-hydroxy-O-methylsterigmatocystin to aflatoxin G1 in Aspergillus parasiticus

In aflatoxin biosynthesis, aflatoxins G(1) (AFG(1)) and B(1) (AFB(1)) are independently produced from a common precursor, O-methylsterigmatocystin (OMST). Recently, 11-hydroxy-O-methylsterigmatocystin (HOMST) was suggested to be a later precursor involved in the conversion of OMST to AFB(1), and conversion of HOMST to AFB(1) was catalyzed by OrdA enzyme. However, the involvement of HOMST in AFG(1) formation has not been determined. In this work, HOMST was prepared by incubating OrdA-expressing yeast with OMST. Feeding Aspergillus parasiticus with HOMST allowed production of AFG(1) as well as AFB(1). In cell-free systems, HOMST was converted to AFG(1) when the microsomal fraction, the cytosolic fraction from A. parasiticus, and yeast expressing A. parasiticus OrdA were added. These results demonstrated (1) HOMST is produced from OMST by OrdA, (2) HOMST is a precursor of AFG(1) as well as AFB(1), and (3) three enzymes, OrdA, CypA, and NadA, and possibly other unknown enzymes are involved in conversion of HOMST to AFG(1).     

Purification and characterization of bovine steroid 21-hydroxylase (P450c21) efficiently expressed in Escherichia coli

Steroid 21-hydroxylase, P450c21, is responsible for the conversion of progesterone and 17alpha-hydroxyprogesterone to their 21-hydroxylated derivatives. P450c21 has been poorly investigated because of difficulty in obtaining sufficient quantities of purified protein. To solve the problem, we have attempted to express the bovine P450c21 in Escherichia coli as a stable form. The N-terminal membrane anchor and basic regions of P450c21 were replaced by the basic region of CYP2C3. The engineered P450c21 was expressed at a level higher than 1.2micromol/L culture (>60mg/L) when coexpressed with molecular chaperones GroES/GroEL. Utilizing three steps of column chromatography, the protein was highly purified to the specific content 16.6nmol/mg (91.2% purity). The purified protein is a monomer in the presence of 1% sodium cholate as determined by gel filtration analysis, suggesting that this membrane anchor-truncated form of P450c21 is more soluble than the native form. The purified enzyme showed typical substrate-binding difference spectra and 21-hydroxylase activities for both progesterone and 17alpha-hydroxyprogesterone. Truncation of the membrane anchor increases solubility of P450c21 facilitating expression of this protein in E. coli yielding sufficient quantities for both biochemical and biophysical studies.     

Chromosomal Locations of Three Human Nuclear Genes (RPSM12, TUFM, and AFG3L1) Specifying Putative Components of the Mitochondrial Gene Expression Apparatus

We have mapped the chromosomal locations of three human nuclear genes for putative components of the apparatus of mitochondrial gene expression, using a combination ofin situhybridization and interspecies hybrid mapping. The genesRPMS12(mitoribosomal protein S12, a conserved protein component of the mitoribosomal accuracy center),TUFM(mitochondrial elongation factor EF-Tu), andAFG3L1(similar to the yeast genesAfg3andRca1involved in the turnover of mistranslated or misfolded mtDNA-encoded polypeptides) were initially characterized by a combination of database sequence analysis, PCR, cloning, and DNA sequencing.RPMS12maps to chromosome 19q13.1, close to the previously mapped gene for autosomal dominant hearing loss DFNA4. TheTUFMgene is located on chromosome 16p11.2, with a putative pseudogene or variant (TUFML) located very close to the centromere of chromosome 17.AFG3L1is located on chromosome 16q24, very close to the telomere. By virtue of their inferred functions in mitochondria, these genes should be regarded as candidates of disorders sharing features with mitochondrial disease syndromes, such as sensorineural deafness, diabetes, and retinopathy.     

Characterization of the L683P mutation of SLC26A9 in Xenopus oocytes

In the present study, we characterized a STAS-domain amino acid mutation of SLC26A9 having a significant impact on ion transport. We focused on the sole conserved L- leucine residue of the STAS domain identified among SLC26 members. We therefore characterized the L683P mutation of SLC26A9 in Xenopus oocytes by monitoring the protein functional expression (two-electrode technique for voltage-clamp analysis) and its presence at the cell membrane (surface protein biotinylation technique). This mutation was found to reduce Cl⁻ transport through SLC26A9 as well as the positive interaction exerted by SLC26A9 on CFTR ion transport activity. The origin of this effect is discussed in the light of the presence of the SLC26A9–L683P mutant at the plasma membrane.     

Angiostrongylus cantonensis: Scanning Electron Microscopic Observations on the Cuticle of Moulting Larvae

Angiostrongylus cantonensis is a parasitic nematode that needs to develop in different hosts in different larval stages. Freshwater snails, such as Pomacea canaliculata, are the intermediate host, and rats are the definitive host. Periodic shedding of the cuticle (moulting) is an important biological process for the survival and development of the parasite in the intermediate and definitive hosts. However, there are few studies on the cuticle alterations between different stages of this parasite. In this study, we observed the ultrastructural appearance and changes of the cuticle of the 2nd/3rd stage larvae (L2/L3) and the 3rd/4th stage larvae (L3/L4) using a scanning electron microscope. We also first divided L2/L3 into late L2 and early L3. The late L2 lacked alae, but possessed a pull-chain-like fissure. Irregular alignment of spherical particles on the cuticle were noted compared to the L3. Alae appeared in the early L3. The old cuticle turned into a thin film-like structure which adhered to the new cuticle, and spherical particles were seen regularly arranged on the surface of this structure. Regular rectangular cavities were found on the surface of L3/L4. The caudal structure of L3/L4 was much larger than that of L3, but caudal inflation, such as seen in L4, was not observed. These results are the first to reveal the ultrastructural changes of the cuticle of A. cantonensis before and after moulting of L2/L3 and L3/L4.     

Helix-Forming tendencies of amino acids depend on their sequence contexts: Tripeptides AFG and FAG show incipient β-bulge formation in their crystal structures

Many of the theoretical methods used for predicting the occurrence of α-helices in peptides are based on the helical preferences of amino acid monomer residues. In order to check whether the helix-forming tendencies are based on helical preferences of monomers only or also on their sequence contexts, we synthesized permuted sequences of the tripeptides GAP, GAV, and GAL that formed crystalline helices with near α-helical conformation. The tripeptides AFG and FAG formed good crystals. The x-ray crystallographic studies of AFG and FAG showed that though they contain the same amino acids as GAF but in different sequences, they do not assume a helical conformation in the solid state. On the other hand, AFG and FAG, which contain the same amino acids but in a different sequence, exhibit nearly the same backbone torsion angles corresponding to an incipient formation of a β-bulge, and exhibit nearly identical unit cells and crystal structures. Based on these results, it appears that the helix-forming tendencies of amino acids depend on the sequence context in which it occurs in a polypeptide. The synthetic peptides AFG (L -Ala-L -Phe-Gly) and FAG (L -Phe-L -Ala-Gly), C₁₄H₁₉N₃O₄, crystallize in the orthorhombic space group P2₁2₁2₁, with a = 5. 232(1), b = 14. 622(2), c = 19. 157(3) Å, D_x = 1.329 g cm^?3, Z = 4, R = 0.041 for 549 reflections for AFG, and with a = 5. 488(2), b = 14.189 (1), c = 18.562(1) Å, D_x = 1.348 g cm^?3, Z = 4, R = 0.038 for 919 reflections for FAG. Unlike the other tripeptides GAF, GGV, GAL, and GAI, the crystals of AFG and FAG do not contain water molecule, and the molecules of AFG or FAG do not show the helical conformation. The torsion angles at the backbone of the peptide are ψ₁ = 144. 5(5)°; ?₂, ψ₂ = ?98.1(6)°, ?65.2(6)° ?₃, ψ₁₃, ψ₃₁ = 154.1(6)°, ?173.6(6)°, 6.9(8)° for AFG; and ψ₁ = 162.6(3)°; ?₂, ψ₂ = ?96.7(4)°, ?46.3(4)°; ?₃, ψ₁₃, ψ₃₁ = 150.1(3)°, ?168.7(3)°, 12.2(5)° for FAG. The conformation angles (? ψ) for residues 2 and 3 for both AFG and FAG show incipient formation of an β-bulge. © 1993 John Wiley & Sons, Inc.     

The requirement of light chain for the surface deposition of the heavy chain of immunoglobulin M

The murine tumor line 70Z/3 resembles a pre-B lymphocyte in containing the heavy chain of IgM (mu) as a cytoplasmic protein in the apparent absence of light chain (L). However, these cells can be induced by lipopolysaccharide to differentiate into a B lymphocyte-like state, containing mu2L2 tetramers as membrane-bound molecules. This is a accompanied by an increase in mu synthesis, the acquisition of complex carbohydrate by mu, and the induction of L chain. We wished to determine which of these events is critical for membrane deposition of mu. We found that uninduced 70Z/3 cells, as well as lipopolysaccharide-uninducible variants, contained a low, constitutive level of membrane bound mu, all of which was found as mu2L2. Dextran sulfate, another inducing agent, apparently caused a redistribution of this pre-existing surface mu without altering the pattern of mu synthesis or processing. One lipopolysaccharide-uninducible variant showed a small subset of surface mu-positive cells, and the proportion of these cells increased with a prolonged induction period. The increase in mu synthesis was nearly normal, but mu did not acquire complex carbohydrate. However, the delayed appearance of surface mu-positive cells was paralleled by a delayed increase in L chain, which occurred only in those cells with mu on their membrane. We concluded that L chain signals the transport of mu to the cell surface.     

Developmentally regulated expression of a cell surface class I nuclease in Leishmania mexicana

Leishmania mexicana, like other trypanosomatid parasites, is a purine auxotroph and must obtain these essential nutrients from its sandfly and mammalian hosts. A single copy gene encoding its unique externally oriented, surface membrane, purine salvage enzyme 3'-nucleotidase/nuclease, was isolated. Structural features of the deduced protein included: an endoplasmic reticulum-directed signal peptide, several conserved class I catalytic and metal co-factor (Zn(2+)) binding domains, transmembrane anchor sequence and a C-terminal cytoplasmic tail. 3'-Nucleotidase/nuclease gene (mRNA) and protein (enzyme activity) expression were examined in three different L. mexicana developmental forms: procyclic promastigotes, metacyclic promastigotes and amastigotes. Results of both approaches demonstrated that the 3'-nucleotidase/nuclease was a stage-specific enzyme, being expressed by promastigote forms (stages restricted to the insect vector), but not by amastigotes (which produce disease in mammalian hosts). Starvation of these parasites for purines resulted in the significant up-regulation of both 3'-nucleotidase/nuclease mRNA and enzyme activity in promastigotes, but not in amastigotes. These results underscore the critical role that the 3'-nucleotidase/nuclease must play in purine salvage during the rapid multiplicative expansion of the parasite population within its insect vector. To our knowledge, the L. mexicana 3'-nucleotidase/nuclease is the first example of a nutrient-induced and developmentally regulated enzyme in any parasitic protozoan.     

Solution structure of ribosomal protein L40E, a unique C4 zinc finger protein encoded by archaeon Sulfolobus solfataricus

The ribosomal protein L40E from archaeon Sulfolobus solfataricus is a component of the 50S ribosomal subunit. L40E is a 56-residue, highly basic protein that contains a C4 zinc finger motif, CRKC_X(10)_CRRC. Homologs are found in both archaea and eukaryotes but are not present in bacteria. Eukaryotic genomes encode L40E as a ubiquitin-fusion protein. L40E was absent from the crystal structure of euryarchaeota 50S ribosomal subunit. Here we report the three-dimensional solution structure of L40E by NMR spectroscopy. The structure of L40E is a three-stranded beta-sheet with a simple beta2beta1beta3 topology. There are two unique characteristics revealed by the structure. First, a large and ordered beta2-beta3 loop twists to pack across the one side of the protein. L40E contains a buried polar cluster comprising Lys19, Lys20, Cys22, Asn29, and Cys36. Second, the surface of L40E is almost entirely positively charged. Ten conserved basic residues are positioned on the two sides of the surface. It is likely that binding of zinc is essential in stabilizing the tertiary structure of L40E to act as a scaffold to create a broad positively charged surface for RNA and/or protein recognition.     

Role of the small GTPase Rac in p22phox-dependent NADPH oxidases

The superoxide-producing phagocyte NADPH oxidase gp91(phox)/Nox2 and the non-phagocytic oxidases Nox1 and Nox3 each form a complex in the membrane with p22(phox), which provides both stabilization and a docking site for organizer proteins. The p22(phox)-complexed Nox2 and Nox1 are dormant on their own, and their activation requires soluble supportive proteins such as a Nox organizer (p47(phox) or Noxo1) and a Nox activator (p67(phox) or Noxa1). The small GTPase Rac directly binds to the activators, and thus plays an essential role in the Nox2-based oxidase containing p47(phox) and p67(phox) or a positive role in Nox1 activity supported by Noxo1 and Noxa1. Although Nox3 complexed with p22(phox) constitutively produce superoxide, the production can be enhanced by supportive proteins. Here we compare the roles of Rac in these p22(phox)-dependent oxidases using the organizer and activator in different combinations. Expression of constitutively active Rac1(Q61L) is essential for activation of the Nox2- or Nox1-based oxidase containing the organizer p47(phox) and either p67(phox) or Noxa1. When these oxidases use Noxo1 as an organizer instead of p47(phox), they produce a small but significant amount of superoxide without expression of Rac1(Q61L), although the production is enhanced by Rac1(Q61L). Thus p47(phox) is likely related to strict dependence on Rac. The Nox3-based oxidase has a similar tendency in the change of the dependence: Rac plays a positive role in Nox3 activation in the presence of p47(phox) and either p67(phox) or Noxa1, whereas Rac fails to upregulate Nox3 activity when p47(phox) is replaced with Noxo1. We also demonstrate that, in the Nox3-based oxidase containing solely p67(phox) as supportive protein, expression of Rac1(Q61L) enhances not only superoxide production but also membrane translocation of p67(phox). Since the enhancements are not observed with a mutant p67(phox) defective in binding to Rac, this GTPase appear to directly recruit p67(phox) to the membrane.     

Comparison of aflatoxin B1 and aflatoxin G1 binding to cellular macromolecules in vitro, in vivo and after peracid oxidation; characterisation of the major nucleic acid adducts.

A comparison between [14C]aflatoxin B1 (AFB1) and [14C]aflatoxin G1 (AFG1) binding to rat liver and kidney cellular macromolecules has shown AFG1-DNA and-ribosomal RNA binding to be lower in both organs. For both mycotoxins more was bound to nucleic acids than to protein. Two hours after intraperitoneal injection (60 microgram/100 g) of [14C] AFB1, 40 ng, 151 ng/mg. Loss of radioactivity bound to liver DNA for both [14C]AFB1 and protein respectively and for [14C]AFG1 the respective figures were 10, 7 and 1 ng/mg. Loss of liver bound radioactivity to DNA for both [14C]AFG1 and [14C]AFG1 appeared to be biphasic indicating that an enzymic DNA repair process may be operating. In vitro binding studies also showed less AFG1 was bound to exogenous DNA after microsomal activation than AFB1. This difference was not a result of differences in the chemical reactivity of the "ultimate" electrophilic species, the respective expoxides, since chemical activation studies using 3-chloroperbenzoic acid showed similar amounts of AFG1 and AFB1 to be converted to the epoxides and to bind to DNA. Studies on the distribution coefficients of the two mycotoxins showed AFB1 to be more lipophilic than AFG1 and this may be an important factor in determining the weaker carcinogenicity of the latter compound. Characterisation of the major AFG1-DNA adduct formed in vitro, in vivo and after peracid oxidation showed it to have the structure trans-9,10-dihydro-9-(7-guanyl)-10-hydroxy-aflatoxin G1. This adduct is similar to that obtained from AFB1 by activation in vivo, in vitro and after peracid oxidation.