7-alkylidenecephalosporin esters as inhibitors of human leukocyte elastase.

A series of 7-alkylidenecephalosporins and 7-vinylidenecephalosporins, as their benzhydryl esters, have been tested as inhibitors of both porcine pancreatic elastase and human leukocyte elastase. Selected 7-alkylidenecephalosporin esters are found to be potent inhibitors of HLE. One category of new inhibitors is the 7-(haloalkylidene)cephalosporins. In contrast to previously reported cephalosporin-based elastase inhibitors, these haloalkylidene cephems show optimum inhibitory activity as sulfides, rather than as sulfones. They are efficient and irreversible inhibitors. A second class of active compounds is represented by the benzhydryl ester 7-(cyanomethylidene)cephalosporin sulfone. In contrast to the activity of these new inhibitors, the benzhydryl ester of the mechanism-based beta-lactamase inhibitor, 7-[(2'-pyridyl)methylidene]-cephalosporin sulfone showed little inhibitory activity as an elastase inhibitor. 7-Vinylidenecephalosporins were also relatively poor inhibitors, although the terminally unsubstituted allene sulfide showed activity as an inhibitor of PPE. A modeling analysis suggests the 7-alkylidene substituents can be readily accommodated in the S1 pocket. A potential mechanism of inhibition is proposed.

n-Propyldiazonium ion alkylates O6 of guanine with rearrangement, but alkylates N-7 without rearrangement.

Treatment of guanosine with n-propylnitrosourea yields 7-n-propylguanine (after acid hydrolysis) but O6-isopropylguanosine. Similarly, injection of [3H]di-n-propylnitrosamine into Sprague--Dawley rats resulted in isolation of 7-n-propylguanine from RNA, but O6-isopropylguanosine. The amounts of the different adducts indicate that attack at both N-7 and O6 of guanine proceed by bimolecular substitution (otherwise significant levels of 7-isopropylguanine should have been seen), but the observed rearrangement indicates that the transition state for reaction at O6 may be much looser than that for reaction at N-7.

PUVA-induced blisters, complement deposition, and damage to the dermoepidermal junction.

We followed the course of 56 patients receiving psoralen plus long-wave ultraviolet light (PUVA) therapy. Nonhemorrhagic blisters developed on clinically normal skin on the limbs of seven patients. Seeming to be related to friction and trauma, the blisters form as a result of damage to the basal and suprabasal layers. Perilesional skin specimens from all blistered patients contained granular deposits of C3 at the dermoepidermal junction, around the upper dermal blood vessels, or at both sites. The average time for initiation and complete formation of suction blisters was measured in 51 patients at different stages during the course of PUVA treatment. Blister separation was in the lamina lucida, with the pemphigoid antigen in the roof while the blister floor contained the lamina densa, laminin, and type IV collagen. This impaired dermoepidermal adhesion was a general phenomenon that occurred in all PUVA-treated patients. The mechanism remains to be determined.

Relative stabilities of nitrenium ions derived from polycyclic aromatic amines. Relationship to mutagenicity.

The relative energetics of arylamine N-hydroxylation and N-O heterolysis (ArNH2----ArNHOH----ArNH+) for condensed systems of two, three and four rings were calculated using semiempirical AM1 molecular orbital theory. The overall thermodynamics of N-hydroxylation were almost insensitive to the structure of the amine while differences in the energetics of nitrenium ion formation varied from 0 to 35 kcal mol-1. Limited correlations between the latter and the experimental TA98 and TA100 mutagenicities of the amines are presented.

Relative stabilities of nitrenium ions derived from heterocyclic amine food carcinogens: relationship to mutagenicity.

The bacterial mutagenicities of a wide variety of complex heteroaromatic amine mutagens and carcinogens present in cooked foods are approximately related to the stabilities of the corresponding nitrenium ions through equations of the kind: log(m) = a delta delta H + b. The stabilities of the nitrenium ions (delta delta H) were computed using the semiempirical AM1 molecular orbital procedure. Parallel calculations of the energies, charge distributions and geometries of simple model compounds provides a qualitative framework within which the stabilities of the nitrenium ions derived from the food carcinogens can be easily understood.

Short-term-toxicity of 4-methyl-1-phenylpentan-2-ol in rats.

Groups of 15 male and 15 female rats were given diets to provide intakes of 0 (control), 10, 40 or 160 mg 4-methyl-1-phenylpentan-2-ol/kg/day for 13 wk. No effects attributable to treatment were seen in the 10-mg/kg/day dose group. At 40 mg/kg/day the only finding was a decrease in serum glucose in males. This was of questionable toxicological significance. At 160 mg/kg/day a reduction in weight gain, probably due to unpalatability, a mild proteinuria in the females, an increased relative liver weight in males, increased caecal weights, a reduction in serum glucose and a lower reticulocyte count were recorded. No changes were observed in the histopathological examination. The no-untoward-effect level established in this study was 10 mg/kg/day.

Long-term toxicity study of carmoisine in rats using animals exposed in utero.

Groups of 114 (control) or 66 (treated) rats of each sex were fed diets providing 0 (control), 100, 400 or 1200 mg carmoisine/kg body weight/day for 9 wk. Within each group the animals were mated monogamously. Treatment continued uninterrupted until the young were randomly selected (where possible one/sex/litter) from each of the litters to provide groups of 90 (control) and 54 (treated) rats of each sex. These received the same treatment as their parents for up to 110 wk for females or 115 wk for males. Apart from coloration of the fur, urine and faeces, treated rats did not differ in appearance or behaviour from the controls. Mortality was not affected by treatment. High-dose groups had reduced body-weight gain compared with that of the controls, despite slightly higher food intakes. Increased water intakes in the same animals accompanied a tendency to excrete larger volumes of urine. Haematological investigations at months 3, 6, 9, 12, 18 and 24, and on all survivors at the end of the study showed no treatment-related effects. Urine studies on 20 rats/sex/group at months 3, 6, 12, 18 and 24 showed no consistent treatment-related changes. Analyses of serum collected at the end of the study demonstrated low glucose concentrations in both sexes of the high-dose group and in intermediate-dose females. A few high-dose males had bladder hyperplasia while some high-dose females had increased incidences of adrenal blood/fibrin cysts or internal hyperplasia/medial hypertrophy of the pancreatic blood vessels. Tumours occurred with a similar distribution and incidence in all groups apart from an increased incidence of adrenal phaeochromocytoma in high-dose males. The incidence seen was well within the background incidence for this relatively common tumour in the same strain of rat under similar conditions. It is concluded that carmoisine is not carcinogenic and that the no-untoward-effect level in this study was 400 mg carmoisine/kg body weight/day.

A 90-day feeding study of lupin (Lupinus angustifolius) flour spiked with lupin alkaloids in the rat.

Three groups of 20 male and 20 female Sprague-Dawley rats were given diets based on lupin (Lupinus angustifolius) flour (55.4 g/100 g diet) that had been spiked to provide dietary concentrations of 250, 1050 or 5050 mg lupin alkaloids/kg diet. A control group of 20 males and 20 females received 50 mg/kg (derived from the background level of alkaloid in lupin flour). The rats were treated for a minimum of 90-98 days. A dose-related reduction in red blood cell count and haematocrit (HCT) occurred in both sexes after 45 days, and the mean cell volume (MCV) was decreased in all the male treatment groups. The reductions in HCT and MCV persisted in the males until termination of the study when decreased haemoglobin levels were also observed in the top-dose males. The relative liver weights of female rats showed a dose-related increase. Altered foci of liver parenchymal cells were seen in five females receiving dietary levels of 5050 mg/kg, in one female fed 250 mg/kg and in one male from each of the 250 mg/kg and 1050 mg/kg treatment groups. No foci were seen in the control group. Basophilic foci are uncommon in young rats suggesting that the low incidence in this study is compound related.

Chronic toxicity/carcinogenicity study of carmine of cochineal in the rat.

Carmine was fed continuously to groups of 54 males and 54 females at dietary levels providing 50, 150 or 500 mg/kg body weight/day for up to 109 wk. As a control, groups of 90 males and 90 females were fed the basal diet for the same period. The rats were derived from parents fed the same dietary levels for 60 days before mating and throughout pregnancy and were thus potentially exposed in utero. There were no adverse effects upon survival, growth or intakes of food and water. No changes associated with treatment were found during the periodic measurement of haematology or renal function, or in the serum chemistry or organ weights at the end of the study. Tumour incidence was not affected, and variations in the distribution of the non-tumour pathology were not considered to be due to treatment. It was concluded that carmine administered to rats in utero and for up to 109 wk is not carcinogenic and that the no-untoward-effect level is 500 mg carmine/kg body weight/day.

A 28-day feeding study with methyl isoeugenol in rats.

Methyl isoeugenol was administered in rodent diet for a minimum of 28 consecutive days to groups of 16 male and 16 female rats (Sprague-Dawley strain) at levels of approximately 30, 100 and 300 mg/kg body weight/day. A further group of 16 male and 16 female rats was given the rodent diet as a control. The administration of methyl isoeugenol in the diet did not adversely affect the growth or general health of the animals or their food intakes. Although high dose animals of both sexes had increased lymphocyte and total white blood cell counts, these are not considered, in isolation, to be an adverse effect of treatment. None of the minor variations observed in the serum chemical analyses or urine analyses is considered to be indicative of a treatment-related toxic effect. An increase in liver weight, adjusted for body weight, was seen in male and female rats receiving 300 mg methyl isoeugenol/kg body weight. Few histopathological abnormalities were observed. Although the incidence of kidney and Harderian gland lesions was higher for high dose animals compared with the controls, the lesions are of a type that occurs spontaneously and are thus not considered to be attributable to treatment with methyl isoeugenol. While the increased liver weight and white blood cell counts of rats given 300 mg methyl isoeugenol/kg body weight may represent effects of treatment, it is not considered that there is any reason to regard these as adverse effects.

A 28-day feeding study with ethyl acetoacetate in rats.

Ethyl acetoacetate encapsulated in gum arabic was administered in rodent diet for a minimum of 28 consecutive days to groups of 16 male and 16 female rats (Sprague-Dawley strain) at levels of approximately 100, 300 and 1000 mg/kg body weight/day. A further group of 16 male and 16 female rats was given rodent diet containing gum arabic as a control. The administration of ethyl acetoacetate in the diet did not adversely affect the growth or general health of the animals or their food intakes. None of the minor variations observed in the haematology, serum chemical analyses or urine analyses are considered to be indicative of a treatment-related toxic effect. Caecal enlargement was seen in male rats treated with the top dose of ethyl acetoacetate, but this was accompanied by a normal histopathology. Few histopathological abnormalities were observed. Proteinaceous casts were found in the bladder of approximately half the male rats given 1000 mg ethyl acetoacetate/kg, and nephrocalcinosis was a common occurrence in female rats in this dose group. Renal function was unimpaired in treated male and female rats, and the histopathological findings are common in the strain of rats chosen for this study. Although the caecal enlargement and the changes in kidney and bladder of rats given 1000 mg ethyl acetoacetate/kg are noted, it is considered that ethyl acetoacetate did not produce treatment-related adverse effects in rats during this study.

Regiochemistry of nucleophilic attack by the guanine 2-amino group at the ring positions of nitrenium ions derived from carcinogenic polycyclic arylamines and nitroarenes: molecular orbital calculations and simple models.

Semiempirical AM1 molecular orbital calculations are used to compute the energetics of addition of the guanine 2-amino group to alternative ring positions of aryl nitrenium ions with the general structure ArNH+, where Ar is the phenyl and various positional isomers of the naphthyl, pyrenyl, and benzo[a]pyrenyl groups. The syn or anti orientation of the NH+ group, and factors akin to classical localization energies, are identified as key components of the differential energetics of addition to alternative ring sites. The regiochemistry predicted by the AM1 method can be qualitatively reproduced using simple HMO calculations that require trivial computational effort and, almost as well, using PMO theory that does not require the use of a computer at all. In the latter approach, the most reactive ring positions are predicted to be those where the nonbonding orbital coefficients, a0r, in the analogous odd alternant hydrocarbons are largest. These results are discussed in relation to the available experimental data for the formation of deoxyguanosin-2-yl adducts when DNA is exposed to presumed nitrenium ion precursors.

Reactions to Quaternium 15, Bronopol and Germall 115 in a standard series.

The incidence of positive patch test reactions to Quaternium 15 has increased by a factor of 3 within a relatively stable population over 2 years, while the incidence of reactions to Bronopol and Germall 115 has remained static. The problem of interpreting an apparent positive reaction to a formaldehyde-releasing preservative in a formaldehyde-sensitive patient is discussed.

The updated electrostatic potential for cytosine completes the qualitative explanations of base alkylation regiochemistry.

The relationship between the structures of different alkylating agents and the relative extents to which they modify the oxygen and nitrogen centers of nucleic acid bases has been discussed in the literature from several points of view, although each effectively attributes the increasing preference for oxygen alkylation to the increasing importance of electrostatic interactions between the reacting moieties. This is in direct contradiction to the published electrostatic potential data for cytosine which indicate the most attractive potential to lie in the vicinity of the 3-nitrogen. However, we have discovered the latter to be an artefact of the use of inadequate levels of theory. When the electrostatic potentials of cytosine are computed using more sophisticated ab initio Hartree-Fock/6-31G* calculations the global minimum does indeed lie in the vicinity of the O2-position as required by the various rationalizations of the alkylation data.

Prediction of nucleoside-carcinogen reactivity. Alkylation of adenine, cytosine, guanine, and thymine and their deoxynucleosides by alkanediazonium ions.

MNDO semiempirical molecular orbital calculations for the SN2 alkylation of nucleic acid bases and deoxynucleosides by the methane-, ethane-, and propanediazonium ions are presented. An approximate correlation is demonstrated between the calculated relative activation enthalpies for attack at alternative base sites and the related experimental quantities for DNA modification by alkylnitrosoureas. The empirically observed shift from N- to O-alkylation with increasing complexity of the alkylating agent is reproduced by the calculations and rationalized by using an extension of a model worked out previously for the analogous reactions of simple nucleophiles. According to this model, the energetics of the related SN1 reactions, while not directly involved, have a profound influence on the SN2 transition-state geometries. For reactions in which the SN1 dissociation is unfavorable the forming bond to the incoming nucleophiles in the related SN2 transition state tends to be short and covalent interactions, which favor N-alkylation, play a significant role. When the SN1 reaction is more facile, the SN2 transition states are "looser" and the covalent interactions correspondingly smaller, leading to an overall shift away from N-alkylation. Consideration of the form of the electrostatic potential around the base, in conjunction with these ideas, provides a detailed explanation of the behavior of electrophiles toward the guanine N2-, 7-, and O6-positions. This model unifies much of the language already used in discussions of nucleic acid regiochemistry. At the same time it is consistent with the geometries and charge distributions in the transition states calculated for the gas-phase reaction processes.

A study of the effects of vigabatrin on the central nervous system and retina of Sprague Dawley and Lister-Hooded rats.

Vigabatrin (gamma-vinyl GABA), an enzyme-activated, irreversible inhibitor of GABA transaminase, was administered orally to albino Sprague Dawley and pigmented Lister-Hooded rats. A dose-dependent retinal lesion characterized histologically by disruption of the outer nuclear layer was observed in the Sprague Dawley rat but not in Lister-Hooded rats, indicating that this alteration is related to the absence of pigment. The lesion is similar to that induced in albino rats by light and certain drugs. In addition, myelin vacuolation of the brain was observed in both rat strains, consistent with the findings of other toxicity studies with vigabatrin. In all cases, the vacuolation was limited to myelinated tracts and resulted from separation of the myelin sheath at the intraperiod line. There was no evidence of demyelination, axonal degeneration or damage to contiguous structures in the affected areas. The vacuolation is histologically similar to that induced in rats by certain other compounds such as isoniazid, hexachlorophene, and triethyltin, but differs in that it is focal in distribution, it is limited to the brain, and is reversible upon cessation of treatment.

Formalin sensitivity and differential staining of mast cells in human dermis.

The characteristics of the human dermal mast cell population with respect to formalin fixation sensitivity, toluidine blue staining and alcian blue/safranin staining were studied. Thirty-seven specimens of normal human skin were bisected. One half was fixed in 10% neutral buffered formalin and the other in Carnoy's fixative. Sections were cut and stained with either toluidine blue or alcian blue/safranin. Significantly more mast cells were visualized with alcian blue/safranin than with toluidine blue. With both stains, only approximately 50% of the mast cells observed in the Carnoy's fixed tissue could be visualized in the formalin-fixed tissue. Alcian blue/safranin staining revealed three patterns of mast cell granule staining: mast cells containing only alcian blue-positive granules, mast cells containing only safranin-positive granules, and mast cells containing a mixture of alcian blue-positive granules and safranin-positive granules. Mast cells containing only alcian blue-positive granules constituted the majority of the dermal mast cell population and 73% of these mast cells were formalin-sensitive. Mast cells containing only safranin-positive granules and those containing a mixture of alcian blue-positive granules and safranin-positive granules showed no evidence of formalin sensitivity. The human dermal mast cell population, therefore, displays heterogeneity with respect to formalin fixation sensitivity and alcian blue/safranin staining. Dermal mast cells were visualized in significantly greater numbers in skin from the head compared with that from the body or limbs.

Langerhans cells in autosomal dominant ichthyosis vulgaris.

Five patients with autosomal dominant ichthyosis vulgaris (ADI) were studied to see whether the abnormal keratinization was associated with disturbances of the appearance or the distribution of epidermal Langerhans cells (LCs). The LCs were identified by ATPase staining and electron microscopy. They were present in normal numbers, were of normal morphology and were in their usual mid-epidermal position. These observations do not support the hypothesis that LCs are involved directly in the process of keratinization.

Quantum chemical characterization of the reactions of guanine with the phenylnitrenium ion.

Density functional calculations at the B3LYP/6-311+G(2d,p)//pBP/DN level predict all cationic adducts combining guanine, at either its N2, O6, N7, or C8 positions, with phenylnitrenium ion, at either its N, 2, or 4 positions, to be lower in energy than the separated reactants. This relative stability of all adducts is preserved after addition of aqueous solvation free energies computed at the SM2 level, although some leveling of the adduct relative energies one to another is predicted. Cations having the lowest relative energies in solution correspond structurally to those adducts most commonly found when guanine reacts with larger, biologically relevant nitrenium ions in vitro and in vivo. One of these, the N-C8 adduct, is stabilized both by a rearomatized phenyl ring and by the operation of an anomeric effect not found in any of the others. On the basis of energetic analysis, direct conversion of an N-N7 cation to an N-C8 cation according to a previously proposed mechanism is unlikely; however, an alternative rearrangement converting a 2-N7 cation to an N-C8 cation via the intermediacy of a five-membered ring may be operative in nitrenium ions with aromatic frameworks better able than phenyl to stabilize endocyclic cationic charge.