Differentiation of interstitial cells and stromal proteins in the secondary septum of early postnatal rat: effect of maternal chronic exposure to whole cigarette smoke

The intention of this investigation was to ascertain the effect of maternal exposure to cigarette smoke on the early postnatal morphogenesis of pulmonary interstitium in offspring. Female rats were chronically exposed to whole cigarette smoke. Offspring of these and control animals were sacrificed at postnatal day 15, and their tissues were prepared for quantitative and qualitative analyses. Results indicate a diminished quantitative representation of parenchymal tissue (P less than 0.01) and a slower pace of secondary septal growth (P less than 0.07) in the experimental lung. Furthermore, a greater cellular volume density (P less than 0.0002) and, inversely, a lesser quantitative representation of extracellular matrix (P less than 0.0002) was ascertained for the experimental septal interstitium. There was proportionately less of elastin substances (P less than 0.009), collagen together with basal laminae (P less than 0.0008), and nonfibrillar, amorphous matrix (P less than 0.02) in the experimental extracellular stroma. Fibrillar collagen and nonfibrillar matrix were represented quantitatively 6.3 times more in the experimental extracellular interstitium than elastin, whereas that ratio for the control tissue was only 4.2. Most experimental interstitial cells (80%) contained numerous lipid globules, which, in contrast, were only occasionally present in control cells (7.3%). Experimental cells, consequently, possessed a larger cross-sectional diameter and a smaller nucleus-to-cytoplasm volume ratio than control cells. These divergent developmental patterns are possibly suggestive of a delayed differentiation of interstitial cells and a modified production to degradation balance of stromal proteins in offspring of animals chronically exposed to whole cigarette smoke.     

Micronucleus induction in V79 cells after direct exposure to whole cigarette smoke

Previous investigations on the effects of cigarette smoke oncultured cells have used mainly smoke condensate dissolved inculture medium. A system has been designed which allows directexposure of cells to fresh cigarette smoke, without an interveninglayer of growth medium between the cells and the smoke. Preliminaryresults have been obtained which demonstrate the viability ofthe system. V79 cells were cultured on porous membranes (Transwell;Costar). During smoke exposure only the lower surface of eachTranswell is supplied with culture medium from the bottom ofthe culture chambers. In this way the cells had direct contactwith the atmosphere at the upper surface and could be exposeddirectly to the test compound. The constructed exposure systemconsists of a smoke generator and an exposure unit containingsix Transwells, the latter contained in an incubator. Cigarettesmoke was generated using a standard 2 s, 35 ml puff once permin. The puff is diluted with conditioned air from the incubatorand injected into the exposure unit. Following exposure of thecells to air only for 3 h there was no effect upon V79 cellviability. However, after exposure to smoke containing between88 and 224 mg/m³ particulate matter, an inhibition of cell proliferationand induction of micronuclei was measured. When a Cambridgefilter pad was placed between the cigarette and the cell exposuresystem to remove particulate matter cell proliferation was alsoreduced and an increased frequency of micronuclei above thecontrol value was measured. ³To whom correspondence should be addressed. Tel: +44 1703 777155; Fax: +44 1703 779856     

Evaluation method for the cytotoxicity of cigarette smoke by in vitro whole smoke exposure

An in vitro whole smoke (WS) exposure method was established to evaluate the toxicological effects of fresh cigarette smoke using the VITROCELL^® system associated with the neutral red uptake (NRU) cytotoxicity assay. The VITROCELL^® system is a newly representative culture and exposure system for in vitro studies of gases or complex mixtures. The impacts of two factors on cytotoxicity measurements of cigarette smoke were investigated using this WS exposure system. The factors include synthetic air exposure and optimal time to perform the NRU assay after smoke exposure. Results showed that synthetic air exposure used in the system did not significantly alter cell survival; 24 h after smoke exposure appeared to be an optimal time-point to assess the cytotoxicity of cigarette smoke. A clear dose–response relationship between smoke exposure and cell viability was demonstrated using this system, and the evaluation method was sensitive to distinguish the differences in smoke-induced cytotoxic effects from different cigarettes. In addition, we tried converting the values of EC₅₀ from WS exposure testing into the values in unit used in total particulate matter (TPM) testing for a purpose of comparison, and the data indicate that the cytotoxicity of smoke measured by WS exposure is greater than that measured by TPM exposure.     

Antinociception induced by chronic exposure of rats to cigarette smoke

To investigate if chronic exposure to cigarette smoke induces analgesia, rats were exposed to concentrated cigarette smoke in an environmental chamber over four successive 5-day blocks (6 h/day), with 2 smoke-free days between blocks. A control group was exposed to room air. Tail flick latencies increased significantly (analgesia) during each smoke exposure block, with a relative decline in analgesia across blocks (tolerance) and a return to control levels during the first three smoke-free interludes while remaining higher after the conclusion of the 4-week exposure period. Mechanical (von Frey) withdrawal thresholds declined over time in smoke-exposed and control groups, with the smoke-exposed group showing significantly lower thresholds. Plasma nicotine reached 95.4 +/- 32 (S.D.) ng/ml at the end of weekly smoke exposure and declined to 44.9 +/- 10.6 ng/ml 24 h after withdrawal. Rats lost weight during smoke exposure and quickly regained weight during smoke-free interludes and at the cessation of smoke exposure. Analgesia may contribute to the initiation of smoking, and rapid reversal of the analgesic effect following acute exposure may contribute to the difficulty in quitting smoking.     

Modified procedure of a direct in vitro exposure system for mammalian cells to whole cigarette smoke.

In vitro biological studies on cigarette smoke have usually been made using either cigarette smoke condensate--obtained by trapping the particulate phase of smoke on a filter, or soluble smoke components--obtained by trapping cigarette smoke in buffer solution. However, these approaches may not truly reflect the physical and chemical condition of freshly generated smoke. Clearly it is important to be able to evaluate the biological effects of fresh smoke on mammalian cells for a better understanding of the potential effects of smoking. The CULTEX technology is a new experimental system for cultivation and exposure techniques enhanced the efficiency of in vitro studies, and allows direct exposure of cells intermittently at the air/liquid interface with ultrafine particles, gases, or mixtures of both which fixedly flows. The CULTEX technology has therefore been modified to evaluate the biological effects of whole cigarette smoke in an in vitro system. The exposure system design was based on a combination of the sedimentation procedure and the CULTEX cultivation technique. After freshly generated smoke was delivered onto cells, the flow was shut off and the medium was slowly removed. In this manner, cells were exposed to both the vapor and particulate phase of smoke efficiently. Cells were maintained in the liquid medium except during the exposure period to maintain the culture conditions and to protect the cells from both the influence of puff pressure and the airflow, which served to remove residual cigarette smoke. The medium was changed at every puff of smoke and so effectively eliminating the possibility of any effects caused by accumulation of soluble cigarette smoke components into the medium. This cycle was repeated and cells were exposed to freshly generated cigarette smoke intermittently.     

Antioxidant gene expression in rat lung after exposure to cigarette smoke.

To investigate the effects of cigarette smoke on the expression of genes encoding intracellular antioxidant species, we exposed rats to whole cigarette smoke or air (control) daily for 1, 2, 7, or 14 days. After sacrifice, RNA was extracted from one lung and expression of mRNA for catalase (CAT), manganese superoxide dismutase (MnSOD), copper-zinc superoxide dismutase (CuZnSOD), glutathione peroxidase (GPX), and metallothionein (MT) was determined by Northern blots and dot blots. The anatomical distribution of expression of these genes was determined by in situ hybridization studies on sections of the contralateral lung. We found that expression of both MnSOD and MT was significantly increased (to levels 70 to 400% greater than in controls) at days 1 and 2 and returned to control levels by day 7. GPX expression was slightly but significantly increased at days 7 and 14 in smoke-exposed animals. CuZnSOD and CAT expression did not change from control levels. In control lungs, MnSOD was expressed in all cell types, with the highest expression seen in bronchial epithelial cells; a notable finding was a mosaic pattern of expression in the bronchial epithelium, with contiguous areas of bronchial epithelium composed of cells expressing MnSOD at high levels (hot spots), compared with the adjacent epithelium. In smoke-exposed lungs, the hot spots became less prominent after 1 and 2 days of exposure to smoke, but after 7 and 14 days the distribution of MnSOD expression was similar in control and smoke-exposed animals. CAT, CuZnSOD, GPX, and MT also showed widespread expression in the lung by in situ hybridization; GPX, CuZnSOD and MT were all most highly expressed in bronchial epithelium, whereas CAT expression levels were similar in all cell types. In contrast to MnSOD, expression of CAT, CuZnSOD, GPX, and MT was uniform within the bronchial epithelium, and the distribution of expression was the same in control and smoke-exposed animals at all time points. We conclude that most of these antioxidant enzymes and scavengers show prominent bronchial expression but that MnSOD shows a unique pattern, with intense hot spots in the epithelium of the small airways. This pattern is similar to the phenomenon of clonal heterogeneity described in other tissues but not previously reported in the lung. We conclude that cigarette smoke, like other forms of oxidant attack, transiently increases expression of MnSOD, and up-regulation of MnSOD expression appears to occur particularly in bronchial epithelial cells, which normally express MnSOD at relatively low levels. MT expression is also transiently increased by smoke whereas GPX expression increases after prolonged (7 to 14 days) exposure to cigarette smoke.     

In vitro micronucleus assay for cigarette smoke using a whole smoke exposure system: A comparison of smoking regimens

Previous studies on the biological assessment of cigarette smoke (CS) mainly focused on the total particulate matter (TPM) collected using a Cambridge filter or gas vapor phase (GVP) bubbled through phosphate-buffered saline (PBS). To study the effects of native CS in vitro, direct exposure methods have been developed. Meanwhile, in vitro micronucleus (MN) assays have been reported to evaluate the mutagenicity of CS.The objective of this research is to investigate the MN-inducing activity of whole smoke (WS) and GVP using a whole smoke exposure system, CULTEX^®, which allows direct exposure of cultured cells to native CS at the air/liquid interface (ALI). CS was generated according to the International Organization for Standardization (ISO; 35 ml, 2 s, once per 60 s) or the Health Canada Intensive (HCI; 55 ml, 2 s, once per 30 s, with complete ventilation block) regimens and Chinese hamster lung (CHL/IU) cells were then exposed to this smoke. Dosages were adjusted according to the amount of smoke entering the actual exposure position. Under both smoking regimens, WS and GVP from 2R4F reference cigarettes induced MN responses. The concept of the dosage and similar dose–response relationships between theoretical and monitored dosage values under the two regimens enabled us to compare the MN-inducing activities of cigarettes in the direct exposure assay, even in the case of various experimental settings or different TPM amounts. MN-inducing activities of 2R4F under the ISO regimen seemed to be higher than those under HCI estimated by the TPM equivalent calculated values.     

Effects of cigarette smoke dose and time after smoke exposure on uptake of asbestos fibers by rat tracheal epithelial cells

We have previously shown that exposure of excised rat tracheal segments to cigarette smoke followed by exposure to a solution of amosite asbestos increases uptake of asbestos fibers compared to exposure to air followed by asbestos. To learn more about the mechanism of smoke-enhanced fiber uptake, we evaluated the effects of amount of smoke and time delay between smoke exposure and asbestos exposure on fiber penetration into the epithelium. To determine whether amount of smoke exposure affected this process, we exposed tracheal segments to 1, 3, or 6 puffs of smoke and subsequently to 5 mg/ml amosite asbestos for 1 h. The segments were then maintained in organ culture for up to 7 d. Asbestos uptake was evaluated by counting fibers in the epithelium by light microscopy. Exposure to increasing numbers of puffs of smoke produced a dose-related increase in fiber uptake at 1 d, 3 d, and 7 d after exposure. To determine whether asbestos exposure needed to occur immediately after smoke exposure for enhanced uptake of fibers to occur, we exposed tracheal segments to 6 puffs of smoke and then delayed exposure to asbestos for 0 (immediate exposure), 3, 18, or 48 h. Tracheas were again maintained in organ culture for up to 7 d after asbestos exposure. Delayed exposure to asbestos after 6 puffs of smoke produced an increase in fiber uptake, even with an interval as long as 48 h between smoke and asbestos exposure; however, the absolute magnitude of fiber uptake was less than that seen with immediate postsmoke asbestos exposure. If catalase was added to the asbestos solution, the smoke-enhanced uptake was abolished, no matter what the time delay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gestational cigarette smoke exposure and hyperthermic enhancement of laryngeal chemoreflex in rat pups

Laryngeal chemoreflex (LCR) apnea occurs in infant mammals of many species in response to water or other liquids in the laryngeal lumen. The apnea can last for many seconds, sometimes leading to dangerous hypoxemia, and has therefore been considered as a possible mechanism in the Sudden Infant Death Syndrome (SIDS). We have found recently that this reflex is markedly prolonged in decerebrate piglets and anesthetized rat pups that are warmed 1–3 °C above their normal body temperatures. We intermittently exposed pregnant rats to cigarette smoke and examined the LCR in their four- to fifteen-day-old offspring under general anesthesia, with and without whole body warming. During warming, pups of gestationally smoke-exposed dams had significantly longer LCR-induced respiratory disruption than similarly warmed control pups. The results may be significant for the pathogenesis and/or prevention of SIDS as maternal cigarette smoking during human pregnancy and heat stress in infants are known risk factors for SIDS.     

Extrapulmonary manifestations of chronic obstructive pulmonary disease in a mouse model of chronic cigarette smoke exposure

Cigarette smoking is the most commonly encountered risk factor for chronic obstructive pulmonary disease (COPD), reflected by irreversible airflow limitation, frequently associated with airspace enlargement and pulmonary inflammation. In addition, COPD has systemic consequences, including systemic inflammation, muscle wasting, and loss of muscle oxidative phenotype. However, the role of smoking in the development of these extrapulmonary manifestations remains rather unexplored. Mice were exposed to cigarette smoke or control air for 6 months. Subsequently, emphysema was assessed by morphometry of lung tissue, and blood cytokine and chemokine levels were determined by a multiplex assay. Soleus, plantaris, gastrocnemius, and tibialis muscles were dissected and weighed. Muscle fiber typing was performed based on I, IIA, IIB, and IIX myosin heavy-chain isoform composition. Lungs of the smoke-exposed animals showed pulmonary inflammation and emphysema. Moreover, circulating levels of primarily proinflammatory proteins, especially TNF-alpha, were elevated after smoke exposure. Despite an attenuated body weight gain, only the soleus showed a tendency toward lower muscle weight after smoke exposure. Oxidative fiber type IIA proportion was significantly reduced in the soleus. Muscle oxidative enzyme activity was slightly reduced after smoke exposure, being most prominent for citrate synthase in the soleus and tibialis. In this mouse model, chronic cigarette smoke exposure resulted in systemic features that closely resemble the early signs of the extrapulmonary manifestations observed in patients with COPD.     

Differentiation ability of rat postnatal dental pulp cells in vitro

The current rapid progression in stem cell research has enhanced our knowledge of dental tissue regeneration. In this study, rat dental pulp cells were isolated and their differentiation ability was evaluated. First, dental pulp cells were obtained from maxillary incisors of male Wistar rats. Immunochemistry by stem cell marker STRO-1 proved the existence of stem cells or progenitors in the isolated cell population. The dissociated cells were then cultured both on smooth surfaces and on three-dimensional (3-D) scaffold materials in medium supplemented with beta-glycerophosphate, dexamethasone, and L-ascorbic acid. Cultures were analyzed by light and scanning electron microscopy and, on proliferation, alkaline phosphatase activity and calcium content were determined and the polymerase chain reaction was performed for dentin sialophosphoprotein, osteocalcin, and collagen type I. These cells showed the ability to differentiate into odontoblast-like cells and produced calcified nodules, which had components similar to dentin. In addition, we found that the "odontogenic" properties of the isolated cells were supported by three-dimensional calcium phosphate and titanium scaffolds equally well.     

Effects of a postnatal exposure to cigarette smoke on hypothalamic catecholamine nerve terminal systems and on neuroendocrine function in the postnatal and adult male rat. Evidence for long-term modulation of anterior pituitary function

Jansson , A., Anderson , K., Bjelke , B., Fuxe , K. & Eneroth , P. 1991. Effects of a postnatal exposure to cigarette smoke on hypothalamic catecholamine nerve terminal systems and on neuroendocrine function in the postnatal and adult male rat. Evidence for long-term modulation of anterior pituitary function. Acta Physiol Scand. 144 , 453–462. Received 10 August 1 991 , accepted 11 August 1991. ISSN 0001–6772. Department of Histology and Neurobiology, Karolinska Instituter, Stockholm, Sweden, Department of Internal Medicine and Unit for Applied Biochemistry, Huddinge Hospital, Huddinge, Sweden. The purpose of this paper was to study the possible long-term effects of postnatal exposure to cigarette smoke. Male Sprague-Dawley rats were exposed to the smoke from 2 cigarettes (Kentucky reference IR-I type) every morning from day 1 after birth for a period of 5 , 10 or 20 days. The rats were decapitated 24 hours ( 5 , 10 and 20 days of exposure), 1 week (20 days of exposure) or 7 months (20 days of exposure) after the last exposure. Using the Falck-Hillarp methodology in combination with quantitative histofluorimetry catecholamine levels and changes in catecholamine utilization (aMT-induced CA fluorescence disappearance) in discrete hypothalamic catecholamine nerve terminal systems were analysed. Serum prolactin, LH, TSH and corticosterone levels were determined by means of radioimmunoassay procedures. In the postnatal period serum LH levels were significantly increased 24 hours after a 10 and 20 day exposure to cigarette smoke. In adult life after a 20–day postnatal exposure to cigarette smoke a highly significant increase was observed in serum prolactin levels, which were unaltered by this exposure when measured in the postnatal period. Twenty-four hours following a 20–day postnatal exposure, catecholamine utilization was increased in the medial palisade zone of the median eminence and substantially reduced in the parvocellular and magnocellular parts of the paraventricular hypothalamic nucleus. One week and 7 months following a 20–day postnatal exposure to cigarette smoke no alterations were observed in catecholamine levels or utilization in various hypothalamic areas including the median eminence. All the above changes were observed in the presence of an unaltered development of body weight. The results indicate that marked but temporary increases in LH secretion occur 24 hours after a postnatal exposure to cigarette smoke, while increase in prolactin secretion only develop in adult life, when the maturational processes of the brain and/or the anterior pituitary gland are completed. Changes in catecholamine levels and utilization are found in discrete hypothalamic nerve terminal networks but do not play a major role in mediating the above changes in anterior pituitary function and are probably the result of a withdrawal phenomenon.     

Changes in neutrophil morphology and morphometry following exposure to cigarette smoke.

Acute cigarette smoking delays neutrophils within the pulmonary circulation in some smokers. Evidence from an in-vitro Micropore filter model of the pulmonary capillaries indicates that this may be due to a smoke induced decrease in cell deformability. In order to determine whether changes in cell shape are associated with the observed decrease in neutrophil deformability following smoke exposure, cell morphology, using scanning electron microscopy, and morphometric measurements, made using transmission electron microscopy, were performed on aliquots of neutrophils harvested from whole blood in non-smoking subjects before and after exposure in vitro to cigarette smoke. Smoke exposure increased the maximum diameter and circumference of neutrophils, without changing their area. There was also a change in the maximum to minimum cell diameter ratio, which indicated that the cells had become less spherical. Scanning electron microscopy showed that smoke exposed cells had developed blebbing of their surface membranes, suggestive of an oxidative injury to the cell membrane rather than the shape changes associated with cell activation. These changes in the morphology and morphometry of smoke exposed neutrophils may contribute to the reduction in cell deformability induced by cigarette smoke.     

Morphology, histochemistry and isozymes of monkey kidney cells during long-term exposure to cigarette smoke.

Monkey kidney cells (Vero) in monolayer culture were exposed to 260 puffs of whole smoke from a mid tar range cigarette over a period of 240 days. Cell morphology was investigated at the light and electron microscopic level. The histochemical characteristics of several hydrolytic enzymes and dehydrogenases were examined, and the isozymes of LDH, MDH and G6PD studied. For all these parameters smoke exposed cells displayed the same features as sham exposed and negative controls. The cells were assessed as non malignant by their failure to form tumours in the nude mouse.     

Squamous cell cancers and cigarette smoke: a matter of exposure

Most of the malignancies associated with tobacco smoke are squamous cell cancers. Others have suggested that squamous cell epithelium has a particular predisposition to tobacco carcinogenesis. However, other malignancies besides squamous cell cancers also have been associated with cigarettes. I hypothesize that the key factor which predisposes an organ or tissue to carcinogenesis by cigarette smoke is exposure to smoke constituents, either directly or indirectly. Squamous cell epithelium has no special biological susceptibility to these carcinogens.     

Effects of a postnatal exposure to cigarette smoke on hypothalamic catecholamine nerve terminal systems and on neuroendocrine function in the postnatal and adult male rat. Evidence for long-term modulation of anterior pituitary function.

The purpose of this paper was to study the possible long-term effects of postnatal exposure to cigarette smoke. Male Sprague-Dawley rats were exposed to the smoke from 2 cigarettes (Kentucky reference IR-1 type) every morning from day 1 after birth for a period of 5, 10 or 20 days. The rats were decapitated 24 hours (5, 10 and 20 days of exposure), 1 week (20 days of exposure) or 7 months (20 days of exposure) after the last exposure. Using the Falck-Hillarp methodology in combination with quantitative histofluorimetry catecholamine levels and changes in catecholamine utilization (alpha MT-induced CA fluorescence disappearance) in discrete hypothalamic catecholamine nerve terminal systems were analysed. Serum prolactin, LH, TSH and corticosterone levels were determined by means of radioimmunoassay procedures. In the postnatal period serum LH levels were significantly increased 24 hours after a 10 and 20 day exposure to cigarette smoke. In adult life after a 20-day postnatal exposure to cigarette smoke a highly significant increase was observed in serum prolactin levels, which were unaltered by this exposure when measured in the postnatal period. Twenty-four hours following a 20-day postnatal exposure, catecholamine utilization was increased in the medial palisade zone of the median eminence and substantially reduced in the parvocellular and magnocellular parts of the paraventricular hypothalamic nucleus. One week and 7 months following a 20-day postnatal exposure to cigarette smoke no alterations were observed in catecholamine levels or utilization in various hypothalamic areas including the median eminence. All the above changes were observed in the presence of an unaltered development of body weight. The results indicate that marked but temporary increases in LH secretion occur 24 hours after a postnatal exposure to cigarette smoke, while increase in prolactin secretion only develop in adult life, when the maturational processes of the brain and/or the anterior pituitary gland are completed. Changes in catecholamine levels and utilization are found in discrete hypothalamic nerve terminal networks but do not play a major role in mediating the above changes in anterior pituitary function and are probably the result of a withdrawal phenomenon.     

Differentiation markers of Sertoli cells and germ cells in fetal and early postnatal human testis

The definition of the temporal sequence of appearance of fetal markers during prenatal and early postnatal development in Sertoli and germ cells may be important for understanding the mechanisms underlying their reexpression in disorders of the adult testis. For this reason, we studied the expression of Sertoli and germ cell markers in 25 human testes spanning a period from 8 gestational weeks to 4 years. Well-characterized antibodies were employed to anti-Müllerian hormone (AMH), cytokeratin 18 (CK18), vimentin (VIM), M2A-antigen (M2A), germ cell alkaline phosphatase (GCAP), and somatic angiotensin-converting enzyme (sACE) on formalin-fixed and microwave-pretreated paraffin sections. In Sertoli cells, AMH and VIM were consistently present. While VIM and CK18 were coexpressed in embryonic testes, CK18 was progressively downregulated and completely absent from the 20th gestational week. M2A was absent or moderately expressed in fetal Sertoli cells but increased during further development. In germ cells, M2A was consistently found in primordial germ cells (PGCs) as well as in M- and T₁-prespermatogonia. In contrast, sACE and GCAP were absent from PGCs but were a distinct feature of late M- and early T₁-prespermatogonia and appeared predominantly between the 18th and the 22nd gestational weeks. Both T₂-prespermatogonia and postnatal prespermatogonia were devoid of any marker. While CK18 represents a differentiation marker for fetal Sertoli cells, M2A, GCAP, and sACE can be used as differentiation markers for the discrimination of different germ cell types during human prespermatogenesis. Because various immunophenotypes reflect distinct differentiation stages, this knowledge may be important for understanding adult testicular pathology.