A review of the safety evidence on recombinant human lactoferrin for use as a food ingredient.

Published studies on the glycosylation, absorption, distribution, metabolism, excretion, and safety outcomes of orally ingested recombinant human lactoferrin (rhLF) were reviewed in the context of unanswered safety questions, including alloimmunization, allergenicity, and immunotoxicity potential of rhLF during repeated exposure. The primary objective was to summarize current safety data of rhLF produced in transgenic host expression systems. Overall, results from animal and human studies showed that rhLF was well tolerated and safe. Animal data showed no significant toxicity-related outcomes among any safety or tolerability endpoints. The no observed adverse effect levels (NOAEL) were at the highest level tested in both iron-desaturated and -saturated forms of rhLF. Although one study reported outcomes of rhLF on immune parameters, no animal studies directly assessed immunogenicity or immunotoxicity from a safety perspective. Data from human studies were primarily reported as adverse events (AE). They showed no or fewer rhLF-related AE compared to control and no evidence of toxicity, dose-limiting toxicities, or changes in iron status in various subpopulations. However, no human studies evaluated the immunomodulatory potential of rhLF as a measure of safety. Following this review, a roadmap outlining preclinical and clinical studies with relevant safety endpoints was developed to address the unanswered safety questions.

Characterization of recombinant human lactoferrin expressed in Komagataella phaffii.

This work presents a thorough characterization of Helaina recombinant human lactoferrin (rhLF, Effera™) expressed in a yeast system at an industrial scale for the first time. Proteomic analysis confirmed that its amino acid sequence is identical to that of native human LF. N-linked glycans were detected at three known glycosylation sites, namely, Asparagines-156, -497, and -642 and they were predominantly oligomannose structures having five to nine mannoses. Helaina rhLF's protein secondary structure was nearly identical to that of human milk lactoferrin (hmLF), as revealed by microfluidic modulation spectroscopy. Results of small-angle X-ray scattering (SAXS) and analytical ultracentrifugation analyses confirmed that, like hmLF, Helaina rhLF displayed well-folded globular structures in solution. Reconstructed solvent envelopes of Helaina rhLF, obtained through the SAXS analysis, demonstrated a remarkable fit with the reported crystalline structure of iron-bound native hmLF. Differential scanning calorimetry investigations into the thermal stability of Helaina rhLF revealed two distinct denaturation temperatures at 68.7 ± 0.9 °C and 91.9 ± 0.5 °C, consistently mirroring denaturation temperatures observed for apo- and holo-hmLF. Overall, Helaina rhLF differed from hmLF in the N-glycans they possessed; nevertheless, the characterization results affirmed that Helaina rhLF was of high purity and exhibited globular structures closely akin to that of hmLF.

Dose Range-Finding Toxicity Study in Rats With Recombinant Human Lactoferrin Produced in Komagataella phaffii.

The oral toxicity of recombinant human lactoferrin (rhLF, Helaina rhLF, Effera™) produced in Komagataella phaffii was investigated in adult Sprague Dawley rats by once daily oral gavage for 14 consecutive days. The study used groups of 3-6 rats/sex/dose. The vehicle control group received sodium citrate buffer, and the test groups received daily doses of 200, 1000, and 2000 mg of rhLF in sodium citrate buffer per kg body weight. Bovine LF at 2000 mg/kg body weight per day was used as a comparative control. Clinical observations, body weight, hematology, clinical chemistry, iron parameters, immunophenotyping, and gross examination at necropsy were used as criteria for detecting the effects of treatment in all groups and to help select dose levels for future toxicology studies. Quantitative LF levels were also analyzed as an indication of bioavailability. Overall, administration of Helaina rhLF by once daily oral gavage for 14 days was well tolerated in rats at levels up to 2000 mg/kg/day, or 57 × Helaina's intended commercial use in adults, and indicating that a high dose of 2000 mg/kg/day is appropriate for future definitive toxicology studies.

Reliable and Accurate Prediction of Single-Residue pKa Values through Free Energy Perturbation Calculations.

Accurate prediction of the pKa's of protein residues is crucial to many applications in biological simulation and drug discovery. Here, we present the use of free energy perturbation (FEP) calculations for the prediction of single-protein residue pKa values. We begin with an initial set of 191 residues with experimentally determined pKa values. To isolate sampling limitations from force field inaccuracies, we develop an algorithm to classify residues whose environments are significantly affected by crystal packing effects. We then report an approach to identify buried histidines that require significant sampling beyond what is achieved in typical FEP calculations. We therefore define a clean data set not requiring algorithms capable of predicting major conformational changes on which other pKa prediction methods can be tested. On this data set, we report an RMSE of 0.76 pKa units for 35 ASP residues, 0.51 pKa units for 44 GLU residues, and 0.67 pKa units for 76 HIS residues.

Insights into the molecular evolution of fertilization mechanism in land plants.

KEY MESSAGE: Comparative genetics and genomics among green plants, including algae, provide deep insights into the evolution of land plant sexual reproduction. Land plants have evolved successive changes during their conquest of the land and innovations in sexual reproduction have played a major role in their terrestrialization. Recent years have seen many revealing dissections of the molecular mechanisms of sexual reproduction and much new genomics data from the land plant lineage, including early diverging land plants, as well as algae. This new knowledge is being integrated to further understand how sexual reproduction in land plants evolved, identifying highly conserved factors and pathways, but also molecular changes that underpinned the emergence of new modes of sexual reproduction. Here, we review recent advances in the knowledge of land plant sexual reproduction from an evolutionary perspective and also revisit the evolution of angiosperm double fertilization.

Gamete Nuclear Migration in Animals and Plants.

The migration of male and female gamete nuclei to each other in the fertilized egg is a prerequisite for the blending of genetic materials and the initiation of the next generation. Interestingly, many differences have been found in the mechanism of gamete nuclear movement among animals and plants. Female to male gamete nuclear movement in animals and brown algae relies on microtubules. By contrast, in flowering plants, the male gamete nucleus is carried to the female gamete nucleus by the filamentous actin cytoskeleton. As techniques have developed from light, electron, fluorescence, immunofluorescence, and confocal microscopy to live-cell time-lapse imaging using fluorescently labeled proteins, details of these differences in gamete nuclear migration have emerged in a wide range of eukaryotes. Especially, gamete nuclear migration in flowering plants such as Arabidopsis thaliana, rice, maize, and tobacco has been further investigated, and showed high conservation of the mechanism, yet, with differences among these species. Here, with an emphasis on recent developments in flowering plants, we survey gamete nuclear migration in different eukaryotic groups and highlight the differences and similarities among species.

Relative Binding Affinity Prediction of Charge-Changing Sequence Mutations with FEP in Protein-Protein Interfaces.

Building on the substantial progress that has been made in using free energy perturbation (FEP) methods to predict the relative binding affinities of small molecule ligands to proteins, we have previously shown that results of similar quality can be obtained in predicting the effect of mutations on the binding affinity of protein-protein complexes. However, these results were restricted to mutations which did not change the net charge of the side chains due to known difficulties with modeling perturbations involving a change in charge in FEP. Various methods have been proposed to address this problem. Here we apply the co-alchemical water approach to study the efficacy of FEP calculations of charge changing mutations at the protein-protein interface for the antibody-gp120 system investigated previously and three additional complexes. We achieve an overall root mean square error of 1.2 kcal/mol on a set of 106 cases involving a change in net charge selected by a simple suitability filter using side-chain predictions and solvent accessible surface area to be relevant to a biologic optimization project. Reasonable, although less precise, results are also obtained for the 44 more challenging mutations that involve buried residues, which may in some cases require substantial reorganization of the local protein structure, which can extend beyond the scope of a typical FEP simulation. We believe that the proposed prediction protocol will be of sufficient efficiency and accuracy to guide protein engineering projects for which optimization and/or maintenance of a high degree of binding affinity is a key objective.

Free Energy Perturbation Calculation of Relative Binding Free Energy between Broadly Neutralizing Antibodies and the gp120 Glycoprotein of HIV-1.

Direct calculation of relative binding affinities between antibodies and antigens is a long-sought goal. However, despite substantial efforts, no generally applicable computational method has been described. Here, we describe a systematic free energy perturbation (FEP) protocol and calculate the binding affinities between the gp120 envelope glycoprotein of HIV-1 and three broadly neutralizing antibodies (bNAbs) of the VRC01 class. The protocol has been adapted from successful studies of small molecules to address the challenges associated with modeling protein-protein interactions. Specifically, we built homology models of the three antibody-gp120 complexes, extended the sampling times for large bulky residues, incorporated the modeling of glycans on the surface of gp120, and utilized continuum solvent-based loop prediction protocols to improve sampling. We present three experimental surface plasmon resonance data sets, in which antibody residues in the antibody/gp120 interface were systematically mutated to alanine. The RMS error in the large set (55 total cases) of FEP tests as compared to these experiments, 0.68kcal/mol, is near experimental accuracy, and it compares favorably with the results obtained from a simpler, empirical methodology. The correlation coefficient for the combined data set including residues with glycan contacts, R2=0.49, should be sufficient to guide the choice of residues for antibody optimization projects, assuming that this level of accuracy can be realized in prospective prediction. More generally, these results are encouraging with regard to the possibility of using an FEP approach to calculate the magnitude of protein-protein binding affinities.

Prediction of Protein-Ligand Binding Poses via a Combination of Induced Fit Docking and Metadynamics Simulations.

Ligand docking is a widely used tool for lead discovery and binding mode prediction based drug discovery. The greatest challenges in docking occur when the receptor significantly reorganizes upon small molecule binding, thereby requiring an induced fit docking (IFD) approach in which the receptor is allowed to move in order to bind to the ligand optimally. IFD methods have had some success but suffer from a lack of reliability. Complementing IFD with all-atom molecular dynamics (MD) is a straightforward solution in principle but not in practice due to the severe time scale limitations of MD. Here we introduce a metadynamics plus IFD strategy for accurate and reliable prediction of the structures of protein-ligand complexes at a practically useful computational cost. Our strategy allows treating this problem in full atomistic detail and in a computationally efficient manner and enhances the predictive power of IFD methods. We significantly increase the accuracy of the underlying IFD protocol across a large data set comprising 42 different ligand-receptor systems. We expect this approach to be of significant value in computationally driven drug design.

Identifying Rare FHB-Resistant Segregants in Intransigent Backcross and F2 Winter Wheat Populations.

Fusarium head blight (FHB), caused mainly by Fusarium graminearum Schwabe [telomorph: Gibberella zeae Schwein.(Petch)] in the US, is one of the most destructive diseases of wheat (Triticum aestivum L. and T. durum L.). Infected grain is usually contaminated with deoxynivalenol (DON), a serious mycotoxin. The challenge in FHB resistance breeding is combining resistance with superior agronomic and quality characteristics. Exotic QTL are widely used to improve FHB resistance. Success depends on the genetic background into which the QTL are introgressed, whether through backcrossing or forward crossing; QTL expression is impossible to predict. In this study four high-yielding soft red winter wheat breeding lines with little or no scab resistance were each crossed to a donor parent (VA01W-476) with resistance alleles at two QTL: Fhb1 (chromosome 3BS) and QFhs.nau-2DL (chromosome 2DL) to generate backcross and F2 progeny. F2 individuals were genotyped and assigned to 4 groups according to presence/ absence of resistance alleles at one or both QTL. The effectiveness of these QTL in reducing FHB rating, incidence, index, severity, Fusarium-damaged kernels (FDK) and DON, in F2-derived lines was assessed over 2 years. Fhb1 showed an average reduction in DON of 17.5%, and conferred significant resistance in 3 of 4 populations. QFhs.nau-2DL reduced DON 6.7% on average and conferred significant resistance in 2 of 4 populations. The combination of Fhb1 and QFhs.nau-2DL resistance reduced DON 25.5% across all populations. Double resistant lines had significantly reduced DON compared to double susceptible lines in 3 populations. Backcross derived progeny were planted in replicated yield trials (2011 and 2012) and in a scab nursery in 2012. Several top yielding lines performed well in the scab nursery, with acceptable DON concentrations, even though the average effect of either QTL in this population was not significant. Population selection is often viewed as an "all or nothing" process: if the average resistance level is insufficient, the population is discarded. These results indicate that it may be possible to find rare segregants which combine scab resistance, superior agronomic performance and acceptable quality even in populations in which the average effect of the QTL is muted or negligible.

Evolutionary multiobjective design of a flexible caudal fin for robotic fish.

Robotic fish accomplish swimming by deforming their bodies or other fin-like appendages. As an emerging class of embedded computing system, robotic fish are anticipated to play an important role in environmental monitoring, inspection of underwater structures, tracking of hazardous wastes and oil spills, and the study of live fish behaviors. While integration of flexible materials (into the fins and/or body) holds the promise of improved swimming performance (in terms of both speed and maneuverability) for these robots, such components also introduce significant design challenges due to the complex material mechanics and hydrodynamic interactions. The problem is further exacerbated by the need for the robots to meet multiple objectives (e.g., both speed and energy efficiency). In this paper, we propose an evolutionary multiobjective optimization approach to the design and control of a robotic fish with a flexible caudal fin. Specifically, we use the NSGA-II algorithm to investigate morphological and control parameter values that optimize swimming speed and power usage. Several evolved fin designs are validated experimentally with a small robotic fish, where fins of different stiffness values and sizes are printed with a multi-material 3D printer. Experimental results confirm the effectiveness of the proposed design approach in balancing the two competing objectives.

Effects of surface pressure on the properties of Langmuir monolayers and interfacial water at the air-water interface.

The effects of surface pressure on the physical properties of Langmuir monolayers of palmitic acid (PA) and dipalmitoylphosphatidic acid (DPPA) at the air/water interface are investigated through molecular dynamics simulations with atomistic force fields. The structure and dynamics of both monolayers and interfacial water are compared across the range of surface pressures at which stable monolayers can form. For PA monolayers at T = 300 K, the untilted condensed phase with a hexagonal lattice structure is found at high surface pressure, while the uniformly tilted condensed phase with a centered rectangular lattice structure is observed at low surface pressure, in agreement with the available experimental data. A state with uniform chain tilt but no periodic spatial ordering is observed for DPPA monolayers on a Na(+)/water subphase at both high and low surface pressures. The hydrophobic acyl chains of both monolayers pack efficiently at all surface pressures, resulting in a very small number of gauche defects. The analysis of the hydrogen-bonding structure/dynamics at the monolayer/water interface indicates that water molecules hydrogen-bonded to the DPPA head groups reorient more slowly than those hydrogen-bonded to the PA head groups, with the orientational dynamics becoming significantly slower at high surface pressure. Possible implications for physicochemical processes taking place on marine aerosols in the atmosphere are discussed.

Capsid protein of cowpea chlorotic mottle virus is a determinant for vector transmission by a beetle.

Cowpea chlorotic mottle virus (CCMV) is a bromovirus transmitted by species of chrysomelid beetles, including the spotted cucumber beetle, Diabrotica undecimpunctata howardii Barber. An experimental system was set up to identify the viral determinant(s) of the beetle transmission of CCMV. Nicotiana clevelandii was selected as an experimental plant host because it supports the replication and accumulation of both CCMV and a second member of the family Bromoviridae, cucumber mosaic virus (CMV). Using a reverse genetic system for CMV, a cDNA copy of the CCMV capsid protein (CP) gene was substituted for that of the CMV CP gene. The resulting 'CMV-hybrid' consisted of wild-type CMV RNA1, RNA2, and a chimeric CMV RNA3 expressing the CCMV structural protein. The CMV-hybrid replicated and formed virions in N. clevelandii; in electron micrographs the hybrid virus was indistinguishable from CCMV. In beetle feeding assays, both CCMV and the CMV-hybrid were transmitted by D. undecimpunctata, while beetle transmission of CMV was not observed. Conversely, only CMV was observed to be transmitted by the aphid Myzus persicae. Surprisingly, the CMV-hybrid was transmitted more efficiently than the parental CCMV, and a virus-induced alteration in beetle feeding behaviour is proposed to account for the difference. These results indicate that the CCMV CP is a viral determinant for beetle vector transmission.

Properties of recombinant Staphylococcus haemolyticus cystathionine beta-lyase (metC) and its potential role in the generation of volatile thiols in axillary malodor.

Enzymes implicated in cysteine and methionine metabolism such as cystathionine beta-lyase (CBL; EC 4.4.1.8), a pyridoxal-5'-phosphate (PLP)-dependent carbon-sulfur lyase, have been shown to play a central role in the generation of sulfur compounds. This work describes the unprecedented cloning and characterization of the metC-cystathionine beta-lyase from the axillary-isolated strain Staphylococcus haemolyticus AX3, in order to determine its activity and its involvement in amino acid biosynthesis, and in the generation of sulfur compounds in human sweat. The gene contains a cysteine/methionine metabolism enzyme pattern, and also a sequence capable to effect beta-elimination. The recombinant enzyme was shown to cleave cystathionine into homocysteine and to convert methionine into methanethiol at low levels. No odor was generated after incubation of the recombinant enzyme with sterile human axillary secretions; sweat components were found to have an inhibitory effect. These results suggest that the generation of sulfur compounds by Staphylococci and the beta-lyase activity in human sweat are mediated by enzymes other than the metC gene or by the concerted activities of more than one enzyme.

Virion stability and aphid vector transmissibility of Cucumber mosaic virus mutants.

The physical stability of virions of Cucumber mosaic virus (CMV) mutants was investigated to determine if relative stability correlated with efficiency of aphid transmission. Virion stability was evaluated by a urea disruption assay and by testing the infectivity of virus following purifications. All viruses were infectious when purified using a low salt buffer without organic solvent, whereas two of seven viruses were less stable and inactivated following purification with a high salt buffer and chloroform. These two viruses were both reassortants derived from the spontaneous transmission-defective mutant CMV-M (F1F2M3 and F1F2M3-L129P). F1F2M3 was relatively unstable, being disrupted between 0 and 1 M urea versus the wild-type CMV-Fny (F1F2M3) that was destabilized at 3-4 M urea. Modifications of F1F2M3 at three amino acid positions (129, 162, 168), singly or in combination, increased the relative stability of virions. A second class of transmission-defective CMVs with engineered mutations in the betaH-betaI surface loop of the CMV-Fny capsid protein (CP) exhibited near wild-type levels of stability. Lastly, a single Pro to Leu substitution at CP position 129 of CMV-Fny (F1F2M3-P129L) conferred the induction of necrosis in tobacco plants and reduced aphid transmissibility, but did not markedly alter the physical stability of virions. Thus, only among CMV-M derivatives harboring the CP mutation of Thr to Ala at position 162 were increases in stability correlated with restoration of transmissibility by the aphid Aphis gossypii.

Identification of the precursor of (S)-3-methyl-3-sulfanylhexan-1-ol, the sulfury malodour of human axilla sweat.

A careful study of human axillary microflora led us to the identification of a new strain of Staphylococcus haemolyticus. The role in axillary malodour formation of this microorganism was compared to those of Corynebacterium xerosis and Staphylococcus epidermidis, upon incubation on sterile human eccrine and apocrine axilla sweat. St. haemolyticus was responsible for the strongest sulfury malodour and the generation of the volatile sulfur compound (VSC) (S)-3-methyl-3-sulfanylhexan-1-ol (3). In this study, we investigated the nonvolatile precursors of VSCs. Human axillary sweat was collected, fractionated and analysed by HPLC/APCI-MS (High-Pressure Liquid Chromatography coupled to Atmospheric Pressure Chemical Ionisation Mass Spectrometry). The precursor of 3 was identified as [1-(2-hydroxyethyl)-1-methylbutyl]-L-cysteinylglycine (Cys-Gly-(S)-conjugate; 12). Because Cys-Gly-(S)-conjugates are key intermediates in the glutathione biodetoxification pathway, other derivatives of 12, specifically glutathione-(S)-conjugate 11 and Cys-(S)-conjugate 13, were prepared. Compounds 11 and 13 were not detected by HPLC/MS of sterile sweat. Synthetic homologues 11, 12, and 13 were incubated with C. xerosis, St. heamolyticus, and St. epidermidis. We observed efficient conversion of precursors 12 and 13 to form VSCs when incubated with St. haemolyticus, with a clear preference for 12. C. xerosis and St. epidermidis were less efficient in cleaving Cys-Gly-(S)-conjugate 12 to form the corresponding thiol 3. Incubation of glutathione-(S)-conjugate 11 never led to the formation of 3 under the experimental conditions employed.

3-Methyl-3-sulfanylhexan-1-ol as a major descriptor for the human axilla-sweat odour profile.

This study sets out to redress the lack of knowledge in the area of volatile sulfur compounds (VSCs) in axillary sweat malodour. Sterile odourless underarm sweat (500 ml) was collected from 30 male volunteers after excessive sweating. Five strains of bacteria, Corynebacterium tuberculostearicum, Corynebacterium minutissimum, Staphylococcus epidermidis, Staphylococcus haemolyticus, and Bacillus licheniformis, were isolated and characterised for their ability to generate an authentic axillary odour from the sweat material collected. As expected, all of the five bacterial strains produced strong sweat odours. Surprisingly, after extensive olfactive evaluation, the strain of Staphylococcus haemolyticus produced the most sulfury sweat character. This strain was then chosen as the change agent for the 500 ml of odourless underarm sweat collected. After bacterial incubation, the 500-ml sample was further processed for GC-olfactometry (GC-O), GC/MS analysis. GC-O of an extract free of organic acids provided three zones of interest. The first was chicken-sulfury, the second zone was onion-like, and the third zone was sweat, clary sage-like. From the third zone, a new impact molecule, (R)- or (S)-3-methyl-3-sulfanylhexan-1-ol, was isolated and identified by GC/MS, MD-GC, and GC AED (atomic emission detector). (S)-3-methyl-3-sulfanylhexan-1-ol was sniff-evaluated upon elution from a chiral GC column and was described as sweat and onion-like; its opposite enantiomer, (R)-3-methyl-3-sulfanylhexan-1-ol, was described as fruity and grapefruit-like. The (S)-form was found to be the major enantiomer (75%).

Investigating the role of polyols in Cladosporium fulvum during growth under hyper-osmotic stress and in planta.

The role of the large amounts of polyols accumulated by the fungal tomato pathogen, Cladosporium fulvum (syn. Fulvia fulva, Cooke) both in planta and in axenic cultures has been examined. Arabinitol and glycerol accumulated in response to hyper-osmotic stress in vitro. Mannitol levels were lower in osmo-stressed mycelium. (13)C NMR spectroscopy indicated that carbon flow from glucose to mannitol was redirected to arabinitol and glycerol in hyper-osmotic conditions. Infected tomato ( Lycopersicon esculentum Mill.) plants contained all three polyols whereas glycerol was the only polyol present in uninfected plants, suggesting that the mannitol and arabinitol were of fungal origin. Substantially higher levels of arabinitol and glycerol were present in infected plants that were subjected to a restricted watering regime compared to fully watered plants. The results suggest that a primary role of fungal arabinitol and glycerol, but not mannitol, is osmoregulation and that water acquisition is an important aspect of pathogenicity.

Diversity Among Isolates of Bean pod mottle virus.

ABSTRACT Isolates of Bean pod mottle virus (BPMV), a member of the genus Comovirus, collected from soybean fields in Kentucky, Virginia, Arkansas, and Iowa were classified into two distinct subgroups, I and II, based on nucleic acid hybridization analysis using cloned cDNA probes to RNA-1 from BPMV strains K-G7 and K-Ha1. Slot blot hybridization analysis using cloned cDNA probes to RNA-2 from the same two strains (K-G7 and K-Ha1), however, revealed that some of the isolates, initially classified as belonging to subgroup I after analysis with RNA-1 probes, are in fact natural reassortants between the two strain subgroups. This was corroborated by nucleotide sequence analysis of full-length cDNA clones of both RNA-1 and RNA-2 from a putative reassortant strain (K-Ho1). These results indicate that BPMV strain diversity is more complex than initially anticipated, and that the use of cloned probes to both genomic RNAs during nucleic acid hybridization analysis is required to unravel the extent of such diversity. In a field plot experiment, BPMV isolates that belong to distinct strain subgroups induced symptoms that varied in severity and in the level of yield losses. In this regard, the reassortant strain K-Ho1 caused the most serious damage compared with four other BPMV isolates tested. Furthermore, the soybean alleles Rsv(1) and Rsv(4), known to confer resistance against Soybean mosaic virus, a member of the genus Potyvirus, did not provide any protection against BPMV. Additionally, we developed a reverse transcription-polymerase chain reaction procedure based on the sequence of a highly conserved region in the capsid polyprotein coding sequence that provides efficient and highly sensitive detection of all BPMV isolates tested, regardless of their strain classification.

Choline Deficiency, Partial Hepatectomy, and Liver Tumors in Rats and Mice.

Choline deficiency (CD) increases susceptability of (he rat liver to a number of hepatocellular carcinogens with a wide diversity of structure and potency. While severe CD results in micronodular cirrhosis and enhanced tumor induction, even a mild deficiency, without cirrhosis is sufficient to result in the increased carcinogenic response. The effects of CD are in part mediated via modulation of microsomal and, possibly, cytosolic enzymes responsible for activation/deactivation of carcinogens. The B6C3F1 hybrid mouse is remarkably sensitive to initiation/promotion of liver tumors by many substances or conditions. Choline deficiency or partial hepatectomy alone, or in concert, markedly enhances liver tumor induction in the absence of any known carcinogen. These data indicate that the liver of this strain of mouse is "initiated" at or shortly after birth and can be promoted by non-carcinogenic substances or conditions.