Angelo Gino Levis

Genetic effects of chromium compounds

Seven different test systems were utilized to investigate the genetic activity of chromium compounds: infidelity of DNA replication in vitro by DNA pol alpha from calf thymus, damage of DNA detected by alkaline elution in treated mammalian cells or in DNA purified and treated in vitro, DNA repair synthesis in mammalian cells in vitro detected by autoradiography or scintillation counting after labelling with [3H]dThd, gene mutations in the Salmonella typhimurium Ames test, gene mutations (6TG resistance) in cultured hamster cells, sister-chromatid exchanges in different rodent cell cultures, and transformation to anchorage-independent growth of hamster cells in vitro (soft-agar assay). Potassium dichromate and chromium chloride were used as water-soluble Cr(VI) and Cr(III) salts. Several reference mutagens (EMS, MMS, MMC, 4NQO) were included in the single tests as positive controls. Cr(VI) was active in all the tested systems, except in the induction of DNA damage and DNA repair synthesis in cultured cells. Cr(III), on the other hand, was absolutely inactive unless a direct interaction with purified DNA was permitted by the test conditions. The relevance of data from the various tests to the understanding of the mechanisms of the genotoxic activity of chromium is discussed. Effects other than the direct interaction of Cr(III) with DNA are inferred, which can cause infidelity of the DNA polymerase functions.

Aging and smoking increase the frequency of sister-chromatid exchanges (SCE) in man.

The frequency of SCE was determined in lymphocytes of 88 healthy human subjects, not occupationally exposed to known genotoxic agents, who were uniformly distributed in several classes of age (from 16 to 70 years), including an equal number of smokers and non-smokers, and of males and females. Our results indicate that the frequency of SCE increases linearly with age and that smoking enhances the frequency of SCE independently of age and sex.

Chromosomal aberrations and sister-chromatid exchanges in Chinese hamster cells treated in vitro with hexavalent chromium compounds

Chinese hamster cells (CHO line) were treated in vitro for 30--39 h with hexavalent chromium compounds (K2Cr2O7 and Na2CrO7), at concentrations ranging from 0.1 to 1.0 microgram of Cr6+ per ml, in medium containing BUdr. Chromosomal aberrations and sister-chromatid exchanges were scored on BUdr-labelled 2nd division metaphases, collected at the end of treatment and stained with Giemsa. Treatment with mitomycin C 0.009--0.030 microgram/ml) was carried out as a control for the responsiveness of the cell system to chromosomal damage. Both chromium compounds induced marked mitotic delays. Chromosomal aberrations were increased about 10-fold by exposure to Cr6+ (1.0 microgram/ml). The principal aberrations observed were single chromatid gaps, breaks and interchanges, whose frequencies increased proportionally to the concentration of chromium. Dicentric chromosomes, isochromatid breaks, chromosome and chromatid rings were also induced. The frequenyc of sister-chromatid exchanges was hardly doubled 30 h after exposure to Cr6+ at 0.3 microgram/ml, whereas it was trebled 39 h after treatment, in the cells whose division cycle had been slowed down by chromium.

Biological monitoring of human exposure to coal tar. Urinary excretion of total polycyclic aromatic hydrocarbons, 1-hydroxypyrene and mutagens in psoriatic patients.

SummaryThree methods for the biological monitoring of human exposure to coal tar were compared. Levels of 1-hydroxypyrene(1-OH PYR), polycyclic aromatic hydrocarbons (PAH) and mutagens (Ames plate incorporation assay using Salmonella typhimurium strain TA98 in the presence of S9 and β-glucuronidase) were determined in urinary samples from psoriatic patients undergoing topical treatment with mineral coal tar. A single sample of urine with a high content of PAH was diluted with urine of non-exposed, non-smoking subjects in order to obtain nine samples with a decreasing content of PAH metabolites. Mutagenicity of the extracts was detectable down to the dilution corresponding to a content in 1-OH PYR of about 50 μg/g creatinine and total PAH of 7 μg/g creatinine. In a second phase the three indicators of exposure to PAH were compared in 16 urinary samples from four psoriatic patients. The total PAH levels determined by the acidic deconjugation/reduction method were confirmed to be nearly always lower than the corresponding levels of 1-OH PYR alone. Most of the extracts were mutagenic, however, some of the samples with a high content in PAH metabolites were not mutagenic. In all the urinary samples analyzed the excretion of 1-OH PYR was markedly greater than in control subjects. 1-OH PYR and urinary mutagenicity levels were well correlated. The present data suggest that both the determination of mutagenicity and 1-OH PYR in urine may be used to monitor occupational exposure to PAH, the latter method being cheaper and of greater specificity and sensitivity.

Clastogenic effects of chromium on human lymphocytes in vitro and in vivo.

Abstract The induction of SCEs and chromosome aberrations in PHA-stimulated human lymphocytes treated in vitro for 70 h with Cr(VI) (potassium dichromate, K 2 Cr 2 O 7 ) or Cr(III) (chromium chloride, CrCl 3 ) was determined. A clear dose-dependent increase of the frequency of SCEs is induced by Cr(VI), a 2.5-fold increase being obtained with 10 −5 M K 2 Cr 2 O 7 . Cr(III) is, on the other hand, absolutely inactive even when used at sub-toxic concentrations (10 −3 M CrCl 3 ). The overall frequency of chromosome aberrations is significantly increased by Cr(VI) above 2.5 × 10 −7 M K 2 Cr 2 O 7 , and chromatid-type aberrations (gaps and breaks) are the most frequent. 100 times higher concentrations of CrCl 3 are required to increase the frequency of chromosome aberrations. A difference of the same order of magnitude is observed for the cytotoxic activity of Cr(VI) and Cr(III), which is characterized by a slowing down of the mitotic cycle reflected by increased 1st-division and decreased 3rd-division metaphases. By treating unstimulated lymphocytes in vitro with Cr(VI) in the G 0 phase, an increase in the frequency of SCEs is produced significantly lower than that obtained on stimulated lymphocytes. Cr(VI) treatments performed at different intervals after stimulation show that the sensitivity of lymphocytes to the induction of SCEs by CR(VI) depends on the phase of the cell cycle, highest frequencies of SCEs being obtained when the cells enter the S phase of DNA replication. The frequency of SCEs in lymphocytes of 12 workers exposed to Cr(VI) (chromic acid fumes in a plating works) is significantly higher than that of 10 control donors. In particular, all the 7 youngest workers, although the most recently engaged in chromium plating, show significantly increased SCE frequencies.

Recent advances in chromium genotoxicity

More than 350 short‐term genotoxicity tests have been performed up to date with Cr compounds, the more recent of which deal with the following aspects: (1) New tests have been developed to evaluate the mutagenicity of Cr, by the introduction of cell strains particularly sensitive to Cr(VI) mutagenicity, and the determination of particular conditions allowing to detect the genetic activity also of Cr(III). (2) Interactions of Cr(VI) and Cr(III) with purified nitrogen bases, polynucleotides, DNA, chromatin, nuclei and whole cells, have led to the identification of specific Cr(III)‐DNA adducts, structural alterations of the genetic material, and effects on the DNA‐repair functions, which can vary in different cell types and tissues. (3) The metabolism of Cr(VI) in mitochondrial, microsomal and soluble cell fractions has been related to inactivation mechanisms, some of which are inducible by Cr(VI) itself. Specific cell components able to reduce Cr(VI) to Cr(III), possibly via genetically active intermediate ...

Persistence of micronuclei in the marine mussel, Mytilus galloprovincialis, after treatment with mitomycin C

The frequency of micronuclei induced by mitomycin C (MMC) in cells of the gill tissue of the marine mussel, Mytilus galloprovincialis Lmk., was determined over a long period (up to 40-52 days) following treatment. Two doses of MMC (0.5 X 10(-7) and 10(-7) M) were tested at 13 degrees C and 23 degrees C, temperatures representative of the winter and summer thermic conditions of the Mediterranean Sea. In all cases, the frequency of micronuclei was significantly increased by MMC and declined after treatment until it reached a plateau level, significantly higher than the control value. This persisted for a very long time. The frequency of micronuclei induced by a second treatment with MMC performed on the 28th day, did not differ significantly from that produced by the first treatment at the same dose. Temperature did not influence the pattern of the described phenomena to a significant extent. The reason for the persistence of an increased frequency of micronuclei is discussed, and a system is proposed for evaluating the genotoxicity of water pollutants present long before sampling.

Effects of trivalent and hexavalent chromium on the physicochemical properties of mammalian cell nucleic acids and synthetic polynucleotides

Abstract The effects of trivalent (chromium chloride) and hexavalent (potassium dichromate) chromium have been studied on the nucleic acids of cultured mammalian cells (BHK hamster fibroblast line), commercial DNA and RNA, and synthetic polynucleotides of known base composition. Modifications of UV absorption spectra and alterations of thermal denaturation and renaturation patterns have been observed by directly treating purified nucleic acids, as well as by examining nucleic acids extracted from cells treated with chromium compounds. Cr(III) interacts with nucleic acid bases, mostly guanine and cytosine, but also with phosphate groups, leading to deprotonation of bases as well as intramolecular cross-links, sandwich complexes between bases and chelation between bases and phosphates. Such interactions destabilize the DNA structure. On the contrary, stabilization of RNA, due to intramolecular metal bonds between nitrogen bases in GC-rich regions, is mainly produced. The kind of interaction of Cr(III) with nucleic acids is not significantly different when intact BHK cells are treated. Cr(VI) interacts similarly with DNA and RNA giving instead different effects when purified nucleic acids or intact cells are treated. Treatment of purified DNA produces breakages in the polynucleotide chain due to the oxidizing power of Cr(VI). In intact cell treatments, changes in the properties of DNA are observed. These could result from the combined action of Cr(III), produced by the intracellular reduction of Cr(VI) and the oxidizing activity of residual Cr(VI). The relevance of Cr(VI) and Cr(III) interactions to the mechanisms of chromium (carcino)genic action is summarized. It is stressed that Cr(VI), if not completely reduced to Cr(III) by extracellular and endoplasmic constituents, can reach the cell nucleus and directly interact with DNA.

Mechanisms of chromium toxicity in mammalian cell cultures

Our observations about the cytotoxic and cytogenetic effects of hexavalent and trivalent chromium compounds in mammalian cells cultured in vitro are reviewed. Additional data concerning the induction of chromosomal aberrations and sister chromatid exchanges, the inhibition of nucleic acid and protein synthesis, the interference with nucleotide metabolism, and the modification of membrane-linked enzyme activity are reported. A possible mechanism of chromium action is proposed.

Cytogenetic damage in workers exposed to ethylene oxide.

Sister-chromatid exchanges (SECs) and chromosomal aberrations (CAs) were detected in the peripheral lymphocytes of 41 sanitary workers exposed to ethylene oxide (EO) in the sterilizing units of 8 hospitals in the Venice Region. The first group (19 workers) was exposed to 10.7 +/- 4.9 ppm EO, expressed as the time-weighted average concentration for an 8-h working day (TWA/8 h conc.), and the second group (22 workers) to 0.35 +/- 0.12 ppm. Each exposed worker was paired with a control of similar age and smoking habits. A highly significant (P less than 0.001) increase in the mean frequency of SCEs was found in the higher exposure group, 14 (74%) exposed subjects having significantly increased levels of SCEs compared to their matched controls. In the lower exposure group, the increase in mean frequency of SCEs was lower, though still significant (P less than 0.05): 7 (33%) exposed subjects had higher and 1 (5%) had a lower SCE level than the matched controls. From the first group, 10 subjects, 7 of whom had increased SCE levels, were reanalysed 12-18 months after their exposure had been lowered or interrupted: in only 2 of them the SCE level was significantly decreased. A statistically significant correlation between SCE frequency and level of EO exposure (TWA/8 h conc.), as well as a multiple correlation between SCE level and EO exposure, smoking and age were found. However, no interaction could be detected between EO exposure and smoking in the induction of SCEs. In controls, SCE frequency was correlated with smoking and age. In the higher exposure group, the number of both chromatid- and chromosome-type aberrations, independent of gaps, was significantly increased, whereas in the lower exposure group only the frequency of chromosome-type aberrations, excluding gaps, was statistically higher than in controls. The level of CAs remained to a great extent unchanged in the 10 subjects re-examined at a later stage after lowering or halting exposure. Taking the group as a whole, the frequency of cells with total CAs was found to be weakly (P = 0.05) correlated with EO exposure, and was not correlated with smoking, age or SCE frequency.