Intrapancreatic delivery of human umbilical cord blood aldehyde dehydrogenase-producing cells promotes islet regeneration.

AIMS/HYPOTHESIS: We sought to investigate the stimulation of islet regeneration by transplanted human umbilical cord blood (UCB) cells purified according to high aldehyde dehydrogenase (ALDH) activity (ALDH(hi)), a conserved characteristic of multiple progenitor lineages. We hypothesised that direct intrapancreatic (iPan) delivery of ALDH(hi) progenitors would augment islet regeneration via timely and localised exposure to islet-regenerative stimuli. METHODS: Cells were purified from UCB based on flow cytometry for low ALDH activity (ALDH(lo)) vs ALDH(hi). UCB ALDH(lo) or ALDH(hi) cells were compared for surface marker expression, as well as haematopoietic, endothelial and multipotent stromal progenitor content in vitro. UCB ALDH(lo) or ALDH(hi) cells were i.v. or iPan injected into streptozotocin-treated non-obese diabetic/severe combined immune-deficient mice temporally monitored for blood glucose, serum insulin and glucose tolerance. Human cell recruitment and survival in the pancreas, insulin content, islet-associated cell proliferation and islet vascularisation were documented in situ. RESULTS: UCB-derived ALDH(hi) cells were highly enriched for haematopoietic and endothelial progenitor frequency, and showed increased expression of progenitor and myeloid cell surface markers. Although i.v. transplantation of ALDH(hi) cells demonstrated low pancreas engraftment and only transient blood glucose lowering capacity, iPan injected ALDH(hi) cells reversed established hyperglycaemia, increased serum insulin and improved the response to a glucose challenge. iPan injected ALDH(hi) cells surrounded damaged islets at early time points and increased islet-associated cell proliferation, resulting in the recovery of beta cell mass. CONCLUSIONS/INTERPRETATION: iPan delivery of UCB ALDH(hi) cells potentiated islet-associated cell proliferation, insulin production and islet revascularisation, resulting in the recovery of host islet function. Elucidation of the progenitor-specific pathways stimulated during islet regeneration may provide new approaches to promote islet expansion during diabetes.

Genetic toxicology assessment of HI-6 dichloride.

The oxime HI-6 dichloride [1-(2 hydroxyiminomethyl -1-pyridino)-3-(4-carbamoyl-1-pyridino)-2-oxapropane dichloride monohydrate] has shown to be a potent reactivator of cholinesterase activity and may have efficacy for the treatment of organophosphate intoxication [SIPRI, 1976; Schenk et al.; Arch Toxicol 36:71-81, 1976]. As part of a preclinical safety assessment program, the genetic toxicology of HI-6 dichloride was evaluated in a series of assays designed to measure induction of gene mutations and chromosomal aberrations. HI-6 dichloride gave negative responses in the Salmonella mutagenicity assay and in the CHO/HGPRT gene mutation assay. Dose-dependent increases in the frequency of chromosomal aberrations were noted when HI-6 dichloride was tested in cultured CHO cells and in cultured human peripheral blood lymphocytes. The mouse lymphoma gene mutation assay, reputed to measure both gene mutations and chromosomal deletions, was negative in the absence of metabolic activation. Depending on the criteria employed, a negative or equivocal response was seen in the presence of rat liver-derived S-9 mix. An in vivo rat bone marrow metaphase assay performed to further investigate the in vitro clastogenic responses was negative. The results from these studies indicate that HI-6 dichloride does not induce gene mutations in vitro; however, it is clastogenic in vitro but does not appear to be clastogenic in vivo.

Induction of mutagenesis and transformation in BALB/c-3T3 clone A31-1 cells by diverse chemical carcinogens.

BALB/c-3T3 cells were employed to examine the genotoxic potential of a variety of known chemical carcinogens. BALB/c-3T3 cells displayed a dose-dependent transformation response to a variety of carcinogens (polycyclic hydrocarbons, methylating agents, ethylating agents, aflatoxin B1 [AFB1], and 4-nitroquinoline-N-oxide [4-NQO]). When the ability of these compounds to induce mutagenesis to resistance to the cardiac glycoside ouabain (OUAR) was examined, we found the short chain alkylating agents to be particularly effective mutagens, causing biologic effects at doses below those necessary to induce a transformation response. In contrast, the polycyclic hydrocarbons which were potent transforming agents were weaker, albeit significant, mutagens for the OUAR locus in this system, while AFB1 was quite weak. Further studies were performed with 5-azacytidine (5-AZA) and the nongenotoxic carcinogen cinnamyl anthranilate (ClN). 5-AZA was a potent transforming agent, but failed to cause mutagenesis. ClN similarly caused in vitro transformation. When a series of eight structurally diverse compounds were examined in both the BALB/c-3T3 and C3H10T1/2 mouse fibroblast transformation systems, the BALB/c-3T3 system was shown to be sensitive to a wide variety of potential carcinogens, whereas the C3H10T1/2 system proved routinely sensitive only to the polycyclic hydrocarbons.

Morphological transformation of BALB/3T3 cells by various procarcinogens in the presence of a rat liver S-9 activation system.

An Aroclor-induced rat hepatic S-9 metabolic activation system was incorporated into the BALB/3T3 cell transformation assay to increase its sensitivity to a wide range of procarcinogens. S-9 was prepared from Aroclor 1254-induced (500 mg/kg) Fischer 344 rats. Cyclophosphamide, dimethylnitrosamine, 2-aminofluorene, and 2-naphthylamine were metabolized to reactive forms capable of inducing both dose-dependent toxicity and morphological transformation of BALB/3T3 cells. Treatments without an exogenous metabolic activation system were nontoxic and nontransforming. Adaptation of this commonly used exogenous metabolic activation system to BALB/3T3 cells will allow detection of the transforming potential of procarcinogens which test negative in a standard assay.

Analysis of spontaneous and induced mutations in transgenic mice using a lambda ZAP/lacI shuttle vector.

A short term, in vivo mutagenesis assay has been developed utilizing a lacl target gene contained within a lambda ZAP shuttle vector which has been incorporated into transgenic mice. Following chemical exposure, the target gene was recovered from mouse genomic DNA by mixing the DNA with in vitro lambda phage packaging extract. Mutations within the lacl target were identified by infecting host E. coli with the packaged phage and plating on indicator plates containing Xgal. Phage plaques with mutations in the lacl appeared blue, while intact phage were colorless. The ratio of blue plaques to colorless plaques is a measure of the mutagenicity of the compound. This system was used to obtain significant induction (up to 74-fold) over background levels for a variety of compounds, including N-ethyl-N-nitrosourea, benzo(a)pyrene (BaP), cyclophosphamide, and methylnitrosourea. Sequence analysis of selected mutant clones derived from this system was accomplished through the use of partial filamentous phage origins which allow rapid transfer of the target gene from phage to plasmid. Sequence analysis of spontaneous mutants derived from the mice primarily found of base substitutions, differing markedly from the previous data for spontaneous mutations in lacl derived from E. coli, where the preponderance of mutations were found at a single site, a repeated tetramer sequence. Upon sequence analysis of BaP derived base substitutions, only transversions were obtained, consistent with the known mechanism of BaP mutagenesis. Use of the well-characterized lacl gene in transgenic mice should allow for extrapolation of the extensive pool of in vitro data to whole animals, as well as provide insight into the tissue specific effects of mutagenic compounds.

Multilaboratory comparison of in vitro tests for chromosome aberrations in CHO and CHL cells tested under the same protocols.

Different test results have been reported for the same chemicals in two in vitro chromosome aberration test systems, CHL cells tested by a Japanese protocol and CHO cells tested by the US National Toxicology Program [Sofuni et al., Mutat Res 241:173-213,1990]. Here, laboratories in Japan, the US and the UK tested 9 such chemicals in CHL and CHO cells using the same protocols and found all 9 positive in both cell types; differences in earlier conclusions with these chemicals were due mainly to test protocol, not to different sensitivities of the cells. The most important protocol difference is sampling time. Chemicals that were negative in the NTP series using a sampling time of 10 to 13 hours often produced positive results when retested here with a 20- to 24-hour sampling time. While positive results were obtained in both cell types, CHL cells sometimes had higher aberration levels and survived at higher doses than CHO cells would tolerate. This may reflect some intrinsic difference in sensitivity but may also be affected by factors such as cell cycle length and culture media (e.g., oxygen scavenging capacity). The collaboration reported here also contributed to a better understanding of scoring aberrations, especially "gaps"; there was good agreement on what types of aberrations should be included in the totals when scoring criteria were clearly defined, for example, many changes classified as "gaps" by the Japanese system were classified as "breaks" in the scoring systems used in the United States and the United Kingdom, and were appropriately included in total aberration counts.

Unusual case of prominent Chiari network trapped in the left atrium.

We describe a case of an unusually prominent Chiari network in a premature neonate who was evaluated with echocardiography. The network, which is an embryologic remnant, was extensive, mobile, and moved in and out of the right ventricle. Later, tissue strands passed the foramen ovale and ultimately became trapped in the left atrium. The differential diagnosis included thrombus, tumor, vegetation, and ruptured chordae.

In vitro and in vivo evaluation of the genotoxic potential of 2-ethyl-1,3-hexanediol.

2-Ethyl-1,3-hexanediol (EHD) has intentional human exposure because of its application to skin as an insect repellent and its use in various skin care products. Genotoxicity studies on EHD were conducted to determine mutagenic and clastogenic potential using in vitro and in vivo test systems. In vitro tests were conducted both with and without an Aroclor-induced, rat-liver S9 metabolic activation system and within a range of cytotoxic to non-cytotoxic doses. EHD did not produce dose-related positive increases in gene mutations in the Salmonella (Ames) test or in the CHO/HGPRT forward mutation test. No statistically significant or dose-related increases in sister chromatid exchanges indicative of DNA damage were produced by EHD in CHO cells. Small but statistically significant increases in chromosome aberrations were produced in CHO cells only in tests with S9 activation. However, no evidence of clastogenicity of EHD was obtained in vivo in a mouse peripheral blood micronucleus test or in 2 rat bone marrow chromosome aberration studies using single or repeated dosing procedures. The overall negative pattern of mutagenic and clastogenic results in the majority of tests conducted suggests that EHD is unlikely to pose significant hazard as a genotoxic agent or to possess carcinogenic initiating activity in animals.

Genotoxicity evaluation of lithium hypochlorite.

Lithium hypochlorite (LiOCl), the pool and spa sanitizer/algicide, was evaluated for genotoxicity in a battery of studies designed to evaluate potential mutagenicity, DNA damage and chromosome aberrations. LiOCl was not mutagenic in the Ames test when tested in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, TA1538 or in the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) mutation assay in Chinese hamster ovary (CHO) cells without metabolic activation. LiOCl did not induce DNA damage in the unscheduled DNA synthesis assay using rat primary hepatocytes. Effects on metaphase chromosomes were evaluated in vitro in CHO cells at 12 and 18 h exposure without S9 and at 12 and 22 h following a 2 h exposure with S9. LiOCl induced a statistically significant increase in chromosome aberrations at the high dose only at both harvest times without S9 and at the late harvest time with S9. There were significant increases in chromosome aberrations at the low dose, low-mid and high doses, but not at the high mid-dose at the early harvest time with S9. However, LiOCl did not increase chromosome aberrations when tested orally in rats at maximally tolerated doses. Bone marrow cells, collected 6, 24 and 48 h after a single oral dose of LiOCl to rats (100, 500, 1000 mg/kg in males; 50, 250, 500 mg/kg in females) showed no increase in the incidence of aberrations. In general, the weight of the evidence indicates that LiOCl is not genotoxic.

Interlaboratory comparison: liver spontaneous mutant frequency from lambda/lacI transgenic mice (Big Blue) (II).

Spontaneous mutant frequency in livers of two transgenic mouse strains, each carrying identical lambda shuttle vectors with a lacI target gene, was evaluated by two laboratories. These studies investigated variability in spontaneous mutant frequency between animals and as a function of the number of phage screened. Liver DNA was independently isolated from 7-11 week old C57BL/6 and B6C3F1 Big Blue transgenic mice. At least 500,000 phage were screened for mutation at lacI for each animal using standardized assay procedures. In the two labs, the C57BL/6 liver spontaneous mutant frequency was 45 +/- 9 x 10(-6) and 41 +/- 7 x 10(-6). The B6C3F1 liver spontaneous mutant frequency was 42 +/- 10 x 10(-6) at one lab and 43 +/- 12 x 10(-6) and 41 +/- 8 x 10(-6) in two trials at the second lab. Mean mutant frequency data from both labs, calculated in increments of 100,000 plaque forming units (pfu) scored for each mouse strain, show stabilized mean mutant frequency and standard deviation after approximately 200,000-300,000 pfu screened. The frequency of spontaneous lacI mutants was reproducible both within and between labs and was comparable between the two transgenic mouse strains.

Intralaboratory optimization and standardization of mutant screening conditions used for a lambda/lacI transgenic mouse mutagenesis assay (I).

A lambda/lacI shuttle vector transgenic mouse mutagenesis assay has been optimized and standardized for reproducible mutant detection. The mutagenic endpoints are blue lacI- phage plaques on a bacterial lawn resulting from the de-repression of beta-galactosidase activity acting on the chromogenic substrate X-gal. Non-mutant lacI phage plaques remain colorless. Factors demonstrated to affect mutant detection include X-gal concentration per assay tray, plaque density per assay tray, pH of plating agar, incubation time at 37 degrees C and the use of a red translucent screening filter over a light source to enhance mutant plaque visibility. In vivo mutant frequencies for liver in untreated animals using standard protocols and internal controls were repeatable in separate experiments using lambda/lacI B6C3F1 mice (4.3 +/- 1.2 x 10(-5) and 4.1 +/- 0.8 x 10(-5)). These studies analyze the use of internal controls to monitor the level of mutant phage plaque detection in a given experiment and evaluate the repeatability of observed mutant frequencies obtained when using standardized procedures.

Baseline and phosphoramide mustard-induced sister-chromatid exchanges in pharmacists handling anti-cancer drugs.

Determinations of baseline and mutagen-induced sister-chromatid exchanges (SCE) have been used as indicators of previous mutagen exposure in several human populations. Mutagen-induced SCE is based on the premise that a genetic outcome may depend not only on a present exposure, but also on a cell's "memory" of previous exposure. The genotoxicity of some anti-cancer drugs including cyclophosphamide (CP) has been studied by determining baseline and mutagen-induced SCE in peripheral blood lymphocytes in treated cancer patients. This study examined the in vivo genotoxic effects of occupational exposure to anti-cancer drug handling by relating baseline and phosphoramide mustard (PM) -induced SCE levels with duration of anti-cancer drug handling as a surrogate for anti-cancer drug exposure dose. The mean baseline SCE for the population was 5.19 +/- 0.17 and was not correlated with duration of drug handling. However, a strong correlation was demonstrated between inducible SCE values and life-time duration of drug handling with r = 0.63 (p less than 0.0001 for low-dose PM challenge (0.1 mg/ml PM) and r = 0.67 (p less than 0.0001) for high-dose PM challenge (0.25 mg/ml PM). A similar relationship was seen for PM-induced SCE and duration of anti-cancer drug handling for the workers' present job with correlations obtained being r = 0.63 (p less than 0.0001) for low-dose PM and r = 0.59 (p less than 0.0001) for high dose PM. The short-lived nature of the baseline SCE lesion is discussed as a limitation in population surveillance studies, as it reflects primarily recent mutagen exposure and persists only for days to weeks after exposure. The induced SCE measure is postulated to provide an integrating dosimeter of remote previous exposure, improving upon the current limitation of the baseline SCE measure and allowing the "unmasking" of previous exposure in a provocative framework.

Evaluation of spontaneous and chemical-induced lacI mutations in germ cells from lambda/lacI transgenic mice.

The spontaneous mutant frequency in germ cells isolated from seminiferous tubules of two lambda/lacI transgenic mouse strains, C57BL/6 and B6C3F1 was evaluated. At least 500 000 phage were screened for mutation at lacI for each animal using standardized assay procedures. The germ cell spontaneous lacI mutant frequency was 17.8 +/- 8.1 x 10(-6) in C57BL/6 mice and 17.0 +/- 10.0 x 10(-6) in B6C3F1 mice. The induction of germ cell mutations by three well characterized alkylating agents were also evaluated in C57BL/6 mice on day 3 after a single dose administration. The lacI mutant frequencies were significantly elevated in transgenic mice dosed with ENU at 150 mg/kg (2-fold increase above control) and iPMS at 200 mg/kg (3-fold increase above control) but not in those receiving MMS at 40 mg/kg. These findings suggest that single dose studies using the lambda/lacI transgenic system may be capable of detecting germ mutations induced by chemicals characterized either by point mutations or small, intragenic deletions but not those characterized by a predominance of multi-locus deletions.

Evaluation of potential genotoxicity of pulsed electric and electromagnetic fields used for bone growth stimulation.

Medical devices emitting pulsed electric and electromagnetic fields have been found to be effective for a number of clinical applications including stimulation of bone and tissue growth. To determine whether pulsed fields of the type used in these clinical applications present a mutagenic hazard, electric and electromagnetic fields at two exposure levels were tested in the Ames test, CHO cell chromosomal aberration assay, BALB/3T3 cell transformation assay and unscheduled DNA synthesis assay in primary rat hepatocytes. For both field types, initial and independent repeat studies were performed for each assay at both clinical and supra clinical doses. In all assays, the results show a lack of cytotoxic, transforming and mutagenic activity. The data suggest that pulsed electric and electromagnetic fields of the type and dose levels used in bone growth stimulation lack mutagenic and transforming activity.

Dose-responsive increase in sister-chromatid exchanges in bone-marrow cells of mice exposed nose-only to whole cigarette smoke.

The effect of whole cigarette smoke exposure on bone-marrow sister-chromatid exchanges (SCEs) was studied in B6C3F1 mice. Animals were exposed nose-only to 10% (v/v) cigarette smoke 5 days/week for 2 weeks. Four dose levels of cigarette smoke (1, 4, 9 and 18 exposures/day) were studied using 2 cigarette types, Kentucky reference 3A1 (3A1) and American Blend (AB). A single exposure represented approximately 1 cigarette. A dose-dependent increase in SCEs was observed for both the 3A1 and AB cigarettes at dose levels which had no effect on bone-marrow cell-replication kinetics. These findings represent the first demonstration of a dose-responsive increase in cigarette smoke-induced SCEs in a rodent model system.

Genetic toxicology of acrylic acid.

Acrylic acid was tested for gene mutations in the in vitro CHO/HGPRT assay, for chromosome aberrations in CHO cells in culture, and for potential to induce unscheduled DNA synthesis in rat hepatocytes in culture. In vivo assays performed included the Drosophila sex-linked recessive lethal assay by both the feeding and injection routes, the in vivo cytogenetic assay in rat bone marrow cells after both a 1-day and 5-day oral dosing regimen, and a dominant lethal assay in mice by both an acute and 5-day dosing regimen. All results were negative (non-mutagenic) except for the in vitro chromosome aberration assay. This latter result is consistent with the previously reported possible clastogenic activity suggested by the results of the mouse lymphoma L5178Y TK locus assay in which a predominance of small-colony mutants was observed (Moore et al., Environmental and Molecular Mutagenesis 1988, 11, 49-63). The rapid clearance of acrylic acid in animals and the weight of evidence of genetic toxicity testing, including negative in vivo data in both somatic and germ cells, indicate a lack of genetic toxicity of acrylic acid in vivo.

Reproductive toxicity, mutagenicity and antigenicity of pamiteplase (genetical recombination).

Pamiteplase (genetical recombination), YM866, is a novel recombinant modified human tissue-type plasminogen activator developed by Yamanouchi Pharmaceutical Co. Ltd., Tokyo, Japan. An intended route of administration in the clinical use of this drug is intravenous administration. We conducted an intravenous fertility and general reproduction studies of this drug in male and female rats and teratology study of this drug in rabbits at the dose levels of 0 (vehicle control), 0.1, 0.3 or 1 mg/kg/day. In the rat, no treatment-related abnormalities were observed up to the maximum dose in parental animals and their offspring. In the teratology study in rabbits, prolonged coagulation time at the injection site was observed at 0.3 mg/kg or more. One death and one abortion occurred at 1 mg/kg on days 22 and 23 of pregnancy, respectively. No toxic effects on the litters were observed up to the maximum dose. Results of evaluation of the mutagenicity of YM866 and its ability to induce chromosome aberrations using the L5178Y TK+/- mouse lymphoma assay, human lymphocyte chromosome aberration assay and the micronucleus assay in mice were negative. Evaluation of the immunogenicity of YM866 by repeated intravenous injection in chimpanzees elicited no confirmed antibody titers.

Virtue and the practice of modern medicine.

Robert Veatch has claimed that virtue theory is not only irrelevant but potentially dangerous in medical ethics. I argue that virtue is a far more prominent factor in contemporary medical practice than Veatch admits. Even if 'stranger medicine' is taken as the norm, proper conduct on the part of physicians depends on certain character traits in order to be maintained consistently over a long period of time and in situations which run counter to the physician's own interests. Right conduct, which Veatch argues is the central moral issue in the physician-patient relationship, is intertwined with certain virtues. Moreover, the virtue of integrity and the concept of a unified life-narrative are especially useful in analyzing an important factor missing in modern medicine. And since medicine relies necessarily on some concept of human flourishing I argue that virtue theory can play a central role in helping to determine the goals of medical practice.