From single to multivariable exposure models to translate climatic and air pollution effects into mortality risk. A customized application to the city of Rome, Italy.

This study presents an approach developed to derive a Delayed-Multivariate Exposure-Response Model (D-MERF) useful to assess the short-term influence of temperature on mortality, accounting also for the effect of air pollution (O3 and PM10). By using Distributed, lag non-linear models (DLNM) we explain how city-specific exposure-response functions are derived for the municipality of Rome, which is taken as an example. The steps illustrated can be replicated to other cities while the statistical model presented here can be further extended to other exposure variables. We derive the mortality relative-risk (RR) curve averaged over the period 2004-2015, which accounts for city-specific climate and pollution conditions. Key aspects of customization are as follows: This study reports the steps followed to derive a combined, multivariate exposure-response model aimed at translating climatic and air pollution effects into mortality risk. Integration of climate and air pollution parameters to derive RR values. A specific interest is devoted to the investigation of delayed effects on mortality in the presence of different exposure factors.

Climate change and air pollution: Translating their interplay into present and future mortality risk for Rome and Milan municipalities.

Heat and cold temperatures associated with exposure to poor air quality lead to increased mortality. Using a generalized linear model with Poisson regression for overdispersion, this study quantifies the natural-caused mortality burden attributable to heat/cold temperatures and PM10 and O3 air pollutants in Rome and Milan, the two most populated Italian cities. We calculate local-specific mortality relative risks (RRs) for the period 2004-2015 considering the overall population and the most vulnerable age category (≥85 years). Combining a regional climate model with a chemistry-transport model under future climate and air pollution scenarios (RCP2.6 and RCP8.5), we then project mortality to 2050. Results show that for historical mortality the burden is much larger for cold than for warm temperatures. RR peaks during wintertime in Milan and summertime in Rome, highlighting the relevance of accounting for the effects of air pollution besides that of climate, in particular PM10 for Milan and O3 for Rome. Overall, Milan reports higher RRs while, in both cities, the elderly appear more susceptible to heat/cold and air pollution events than the average population. Two counterbalancing effects shape mortality in the future: an increase associated with higher and more frequent warmer daily temperatures - especially in the case of climate inaction - and a decrease due to declining cold-mortality burden. The outcomes highlight the urgent need to adopt more stringent and integrated climate and air quality policies to reduce the temperature and air pollution combined effects on health.

Air pollution impact assessment on agroecosystem and human health characterisation in the area surrounding the industrial settlement of Milazzo (Italy): a multidisciplinary approach.

In order to evaluate the impact of atmospheric pollutants emitted by the industrial settlement of Milazzo (Italy) on agriculture, sulphur dioxide and ozone levels in air were monitored and the data were used to estimate yield losses of the most widespread cultures. Trace element concentrations in crops and soils were also detected and metabolic profiles of soil microbial communities were considered. Vibrio fischeri test was used to appraise airborne pollutant ecotoxicity and epidemiological studies on causes of death distribution were carried out to characterize health state of people living in the area. All the sampling points were selected in farms on the basis of a theoretical meteo-diffusive model of industrial air pollutants. Experimental SO2 and O3 values mainly exceeded the threshold established by Italian and EU regulations to protect vegetation and they correspond to estimated significant crop losses. Conversely toxic element residues in soils and in agroalimentary products were generally lower than the fixed values. SO2 and O3 concentrations, toxic element contents and ecotoxicity levels of airborne pollutants were not related only to industrial site emissions, while the fluctuations on metabolic profiles of soil microbial communities seem to agree with the predicted deposition of xenobiotic compounds from the industrial plants. The epidemiological study evidenced a better health state of populations living in the investigated area than in the Messina province and the Sicily region but, inside the area, males living in the municipalities closest to the industrial settlement exhibited a worst health state than those in the very far ones.

Retrospective mortality cohort study of Italian workers compensated for silicosis.

OBJECTIVES: To estimate cause specific mortality in a large cohort of Italian workers compensated for silicosis. METHODS: The cohort included 14 929 subjects (14,098 men and 831 women) compensated for silicosis between 1946 and 1979, alive on 1 January 1980, and resident in Tuscany (a region of central Italy with 3,547,000 inhabitants). Mortality follow up ranged from 1980 to 1999. Vital status and the causes of death were determined by linkage with the regional mortality registry and with the national mortality database. The cohort mortality rates were compared to the rates of the local reference population. SMRs and their 95% confidence intervals were computed assuming a Poisson distribution of the observed deaths. Specific SMR analyses were performed according to the level of disability, the year of compensation assignment, and the job type. RESULTS: A significant excess mortality was observed in male silicotics for cancer of the lung, trachea, and bronchus and cancer of the liver, respiratory diseases (silicosis, asbestosis, antracosilicosis, and other pneumoconiosis), and for tubercolosis. Statistically significant mortality excess was observed in female silicotics for respiratory diseases (specifically silicosis and other pneumoconiosis) and tuberculosis. Analyses for period of compensation assignment showed a twofold increased SMR for biliary tract cancer among female workers and for liver cancer among male workers compensated before 1970. CONCLUSIONS: The excess mortality from respiratory tract cancers and respiratory tract diseases detected in Italian compensated silicotics are in agreement with previous epidemiological studies. Although the twofold increased risk for liver cancer among males is suggestive of a possible association with silica dust exposure, the finding needs to be confirmed.

[Distribution of causes of death in communities with different urbanization levels]. / Distribuzione delle cause di morte in comuni a diverso grado di urbanizzazione.

The aim of the present work was to compare mortality data during 1980-94 (24 causes of death for males and 25 for females) in populations living in municipalities with different urbanization levels of seven Italian regions (Piemonte, Lombardia, Emilia Romagna, Marche, Lazio, Campania and Sicilia). Urbanization levels were established following the classification of the Italian Institute of Statistics (ISTAT, Classificazione dei comuni secondo le caratteristiche urbane e rurali, 1986). A quite uniform configuration of causes of death with characteristic excesses in the urban environment was evident. The only exception was represented by the male population in Lombardia, probably due to the prevalence of occupational exposure. The urban excesses included: all tumors (also for the population < 64 year old), trachea, bronchi and lung tumors (also < 64 year old people), colorectal cancer, bladder cancer, male liver cancer and female breast cancer. Mortality due to stomach cancer and, only in males, to larynx cancer was lower in urban than in the other types areas, except the Southern regions. Concerning some non tumoral pathologies a more dyshomogeneous situation was observed, with a prevalence of urban defects in Piemonte and Lombardia, of urban excesses in the Southern regions and of both excesses and defects in Emilia Romagna and the Central regions. The "semi-rural" and "rural" types municipalities resulted quite similar, with the exception of Campania and Lazio. In fact, in these two regions a greater association with stomach cancer and lower mortality for all other tumors compared to the urban and, at a lesser extent, to the semi-urban municipalities was found. However, the lower tumor mortality was partly balanced by some other causes of death, again with the exception of Campania and at a lesser extent of Sicilia. On the basis of the causes of death comparisons, the semi-urban municipalities seem to represent a transition type between the urban and the rural ones.

Effects of heat on mouse spermatogenesis monitored by flow cytometry.

The effects of heat on mouse spermatogenesis have been determined using both testis weight and flow cytometrically determined DNA content distribution as experimental end-points. Temperatures of 38-42 degrees C and exposure times of 20-60 min have been tested. The results concerning the testis weight substantially confirm those reported by other authors (Hand et al. 1979). The measurement of DNA content distributions shows a relatively higher depletion, 14 days after treatment, of the cytometric compartment containing elongated spermatids in respect to that containing round spermatids. The analysis of the cytotoxic effects, monitored 14 vs. 28 days after treatment, as a function of the exposure time at a given temperature, or of the temperature for a fixed exposure time, indicates that, in the course of spermatogenesis, late spermatocytes are more sensitive to heat than differentiated spermatogonia. Following the approach based on flow cytometry, the effect of exposures as low as 20 min at 38 degrees C can be appreciated.

Effects of L-acetylcarnitine (LAC) on the post-injury recovery of mouse spermatogenesis monitored by flow cytometry. 1. Recovery after X-irradiation.

L-acetylcarnitine plays a key role in sperm metabolism and in the whole spermatogenetic process. In the present work, the influence of L-acetylcarnitine, administered i.p. (100 mg/kg body weight), on the recovery processes of mouse spermatogenesis after local acute irradiation with 10 Gy X-rays has been investigated. The effects were monitored 28, 35, 40, 45, 50, 55, and 60 days after irradiation by flow cytometric analysis of cellular DNA content. In the LAC-treated animals, the fraction of tetraploid cells is higher at 28 (p less than 0.05) and 45 days (p less than 0.02). Corresponding with the timing of the stages of murine spermatogenesis, the round spermatid fraction is higher at 45 days (p less than 0.1) and the elongated spermatid fraction is higher at 50 days (p less than 0.1) after irradiation. In addition, the LAC-treated animals show a faster recovery throughout the maturation process, from tetraploid to round and elongated spermatids. These results indicate that the presence of exogenous LAC could enhance the recovery of spermatogonial cells.

Automatic analysis of flow cytometric DNA histograms from irradiated mouse male germ cells.

An automatic procedure for recovering the DNA content distribution of mouse irradiated testis cells from flow cytometric histograms is presented. First, a suitable mathematical model is developed, to represent the pattern of DNA content and fluorescence distribution in the sample. Then a parameter estimation procedure, based on the maximum likelihood approach, is constructed by means of an optimization technique. This procedure has been applied to a set of DNA histograms relative to different doses of 0.4-MeV neutrons and to different time intervals after irradiation. In each case, a good agreement between the measured histograms and the corresponding fits has been obtained. The results indicate that the proposed method for the quantitative analysis of germ cell DNA histograms can be usefully applied to the study of the cytotoxic and mutagenic action of agents of toxicological interest such as ionizing radiations.

Cytotoxic effects of benzene on mouse germ cells determined by flow cytometry.

Flow cytometric (FCM) DNA content measurements were performed on testicular monocellular suspensions obtained from mice exposed per os to 0, 1, 2, 4, 6, and 7 ml/kg body weight of benzene in order to investigate its cytotoxic action on germ cells. The effects of benzene were measured 7, 14, 21, 28, and 70 d after treatment. Benzene had no effect on testis weight, but FCM analysis showed the relative percentages of some cell subpopulations (tetraploid and haploid cells) to be different from the control pattern, indicating the occurrence of some cytotoxic damage to differentiating spermatogonia. These data demonstrate that spermatogenesis is sensitive to benzene single exposures as evidenced by an altered cell ratio of testicular cell types.

Flow cytometric analysis of the effects of 0.4 MeV fission neutrons on mouse spermatogenesis.

(C57Bl/Cne X C3H/Cne)F1 male mice were irradiated with single acute doses of 0.4 MeV neutrons ranging from 0.05 to 2 Gy, and testis cell suspensions were prepared for cytometric analysis of the DNA content 2-70 days after irradiation. Various cell subpopulations could be identified in the control histogram including mature and immature spermatids, diploid spermatogonia and spermatocytes, tetraploid cells and cells in the S-phase. Variations in the relative proportions of different cell types were detected at each dose and time, reflecting lethal damage induced on specific spermatogenetic stages. The reduction of the number of elongated spermatids 28 days after irradiation was shown to be a particularly sensitive parameter for the cytometrical assessment of the radiosensitivity of differentiating gonia. A D0 value of 0.13 Gy was calculated and compared with data obtained after X-irradiation, using the same experimental protocol. In the latter case a biphasic curve was obtained over the dose range from 0.25 to 10 Gy, possibly reflecting the existence of some cell population heterogeneity. RBE values were estimated at different neutron doses relative to the radiosensitive component of the X-ray curve, and ranged from 3.3 to 4, in agreement with data in the literature. Genotoxic effects were monitored 7 days after irradiation by a dose-dependent increase of the coefficient of variation (CV) values of the round spermatid peak, reflecting the induction of numerical and structural chromosome aberrations, and 14 or 21 days after irradiation by the detection of diploid elongated spermatids, probably arising from a radiation-induced complete failure of the first or second meiotic division.

Flow cytometrically determined DNA content of breast carcinoma and benign lesions: correlations with histopathological parameters.

The relative DNA content of cellular samples from 54 patients affected by breast carcinomas and 20 affected by benign breast lesions (including 11 fibroadenomas) was measured by flow cytometry. All normal tissue samples and 17/20 (85%) specimens from benign lesions exhibited a cytometrically diploid DNA distribution, 3/20 (15%) benign lesions an abnormal DNA content, and 35/54 (65%) carcinomas at least one aneuploid cell subpopulation. Furthermore, 9/54 (17%) tumors were characterized by the presence of more than one aneuploid cell subpopulation. The results also indicate that flow cytometry can be used to recognize lymph nodes infiltrated by aneuploid cells. Statistically significant correlations were evidenced between the occurrence of aneuploidy or the ploidy level measured as DNA index and the nodal infiltration status. The percentage of S cells can also be extracted from DNA content distribution histograms. Statistically significant differences (p less than 0.01) were also observed for the percentage of S cells between normal tissues (6.2 +/- 3.2 SD) and benign lesions (11.1 +/- 6.6 SD), normal tissues (6.2 +/- 3.2 SD) and aneuploid tumors (19.7 +/- 10.3 SD), benign lesions (11.1 +/- 6.6 SD) and aneuploid tumors (19.7 +/- 10.3 SD), and diploid (7.9 +/- 4.0 SD) and aneuploid tumors (19.7 +/- 10.3 SD).

Non-small-cell lung carcinoma: tumor characterization on the basis of flow cytometrically determined cellular heterogeneity.

Some 150 tumor specimens from 49 patients with non-small-cell carcinoma of the lung (23 epidermoid, 14 adenocarcinoma, 12 large-cell carcinoma) and three with nonneoplastic lung disease were analysed for cellular DNA content by flow cytometry. Monodispersed cells were stained with ethidium bromide and mithramycin. Normal specimens and samples from patients with nonneoplastic disease constantly yielded a single cell population with diploid DNA content. Twenty of 23 epidermoid carcinomas exhibited one or more than one aneuploid subpopulation. Ten of 12 large-cell carcinomas were characterized by one aneuploid clone and 2/12 by two aneuploid clones. Adenocarcinoma exhibited multiclonal cell subpopulations (one to five aneuploid clones). Further information has been obtained on the differential presence of clones in various tumor areas and in infiltrated lymph nodes. These tumors appear characterized by a remarkable degree of cellular heterogeneity. The cytometric ploidy level(s) and the cell population multiclonal structure yield, in comparison with, and in addition to, pathology, indications of possible clinical interest. A correlation between the clonal DNA content and a prognostic parameter such as the tumor mass doubling time has been demonstrated.