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Biomedical subjects

R M Tyrrell

Publications and source records attributed to R M Tyrrell.

At least 55 records · Page 3Linked to original sources
PubMed1996

UV activation of mammalian stress proteins.

Ultraviolet radiation may be divided into the non-solar UVC region, the solar UVB (290-320 nm) region which is strongly absorbed by nucleic acids, and the solar UVA (320-380 nm) region which is less strongly absorbed by nucleic acids and proteins but causes a variety of oxidative events. As a consequence of these different properties, UVC/UVB radiations induce an array of stress proteins quite distinct from those induced by UVA radiations. Although many studies with UVC and UVB radiations involve lethal doses, it is clear that these radiations have the property of mimicking growth factor responses and stimulate various signal transduction pathways that lead to gene activation including transcriptional activation of the jun and fos proto-oncogenes. Furthermore, UVB irradiation of skin, at physiologically relevant doses can increase the levels of various stress proteins including ornithine decarboxylase, various cytokines, the p53 tumor suppressor protein and to a limited extent, nuclear oncogene products. Non-cytoxic exposures of UVA radiation can lead to the up-regulation of several genes including collagenase, heme oxygenase 1, a specific protein phosphatase (CL 100) and phospholipases. At least for heme oxygenase 1, there is evidence that the alteration may be involved in a pathway of defense against oxidative stress. However, much information is lacking in the quest to build up a complete picture of the physiological and pathological significance of the many UV inducible stress responses reported.

Animals↗
PubMed1995

UVA radiation-induced oxidative damage to lipids and proteins in vitro and in human skin fibroblasts is dependent on iron and singlet oxygen.

This study describes the damage that occurs to lipids and proteins that have been irradiated in vitro or in human skin fibroblasts with physiological doses of UVA radiation. Thiobarbituric acid-reactive species were formed from phosphatidylcholine after UVA radiation in vitro. By using iron chelators, this process was shown to involve iron. Ferric iron associated with potential physiological chelators was reduced by UVA radiation, but iron within ferritin was not. By enhancing the half life-time with deuterium oxide or by using scavengers, singlet oxygen was also shown to be involved in the UVA radiation-dependent peroxidation of phosphatidylcholine. UVA radiation-generated singlet oxygen reacted with phosphatidylcholine to form lipid hydroperoxides, and the breakdown of these hydroperoxides to thiobarbituric acid-reactive species was dependent on iron. We have shown that iron and singlet oxygen are also involved in the UVA radiation-dependent formation of thiobarbituric acid-reactive species in human skin fibroblasts, and we propose that a similar concerted effect of iron and singlet oxygen is involved in UVA radiation-dependent damage to fibroblast lipids. Sulphydryl groups of bovine serum albumin and human gamma-globulin were oxidised upon UVA irradiation in vitro. The use of scavengers and deuterium oxide showed that UVA radiation-dependent sulphydryl oxidation was dependent on singlet oxygen. By adding or chelating iron, UVA radiation-dependent oxidation of sulphydryl groups of bovine serum albumin and human gamma-globulin was shown to be iron-dependent. The use of catalase and hydroxyl radical scavengers demonstrated that hydrogen peroxide, but not the hydroxyl radical, was involved. The oxidation of sulphydryl groups of proteins in human skin fibroblasts that occurs as a result of UVA irradiation was also shown to involve iron, singlet oxygen, and hydrogen peroxide. We conclude that iron, singlet oxygen, and hydrogen peroxide are important redox active species involved in the deleterious effects of UVA radiation on lipids and proteins of human skin cells.

Dose-Response Relationship, Radiation↗
PubMed1995

Biological dosimetry and action spectra.

In view of the current renewed interest in biological dosimeters, data obtained in the mid-1970s with the UV-sensitive Bacillus subtilis spore UVSSP both in natural sunlight in Rio de Janeiro and with defined monochromatic radiation is reproduced and reconsidered. The crucial issue of the correspondence of the wavelength dependence of the dosimeter response and the biological effect of interest is discussed with reference to end points such as cytotoxicity and melanoma and non-melanoma skin cancer induction. The necessity of taking into account UVA as well as UVB effects in the overall biological effectiveness of sunlight is evaluated and discussed. Gene activation is proposed as a basis for developing sensitive biological markers that can differentiate between UVA and UVB effects and also be used to determine UV penetration into target tissue.

Animals↗
PubMed1995

Ultraviolet radiation and free radical damage to skin.

Solar UVB (290-320 nm) and particularly UVA (320-380 nm) radiations have a capacity to generate reactive chemical species, including free radicals, in cells. These intermediates have been shown to be involved in various biological effects in cultured human skin cells (e.g. cell death) and skin (e.g. erythema). Endogenous glutathione is a critical molecule in protection against the cytotoxic effects of both wavelength ranges. Although there is evidence from cellular studies for the involvement of an oxidative component of UVC/UVB radiations in activation of several genes, the doses used are generally extremely cytotoxic and could cause aberrant signalling. Genes activated by sublethal doses of UVA radiations (e.g. haem oxygenase 1 and the CL100 phosphatase) are clearly redox regulated. The strong induction of haem oxygenase 1 in human fibroblasts has been implicated in an adaptive response to oxidative membrane damage that involves increased synthesis of the iron storage protein, ferritin.

Gene Expression Regulation↗
PubMed1995

Activation of NF-kappa B in human skin fibroblasts by the oxidative stress generated by UVA radiation.

We have examined the role of the nucleus and the membrane in the activation of nuclear factor (NF)-kappa B by oxidant stress generated via the UVA (320-380 nm) component of solar radiation. Nuclear extracts from human skin fibroblasts that had been irradiated with UVA at doses that caused little DNA damage contained activated NF-kappa B that bound to its recognition sequence in DNA. The UVA radiation-dependent activation of NF-kappa B in enucleated cells confirmed that the nucleus was not involved. On the other hand, UVA radiation-dependent activation of NF-kappa B appeared to be correlated with membrane damage, and activation could be prevented by alpha-tocopherol and butylated hydroxytoluene, agents that inhibited UVA radiation-dependent peroxidation of cell membrane lipids. The activation of NF-kappa B by the DNA damaging agents UVC (200-290 nm) and UVB (290-320 nm) radiation also only occurred at doses where significant membrane damage was induced, and, overall, activation was not correlated with the relative levels of DNA damage induced by UVC/UVB and UVA radiations. We conclude that the oxidative modification of membrane components may be an important factor to consider in the UV radiation-dependent activation of NF-kappa B over all wavelength ranges examined.

Fibroblasts↗
PubMed1995

Two genes contribute to different extents to the heme oxygenase enzyme activity measured in cultured human skin fibroblasts and keratinocytes: implications for protection against oxidant stress.

Activation of expression of the heme oxygenase (HO) gene appears to be involved in a cellular defense system in mammalian cells. We now demonstrate that while HO-1 mRNA levels are strongly inducible in dermal fibroblasts they are barely inducible in human epidermal keratinocytes following oxidative stress (UVA radiation and hydrogen peroxide). Paralleling this result was the observation that HO-2 mRNA levels were low in dermal fibroblasts but were high in epidermal keratinocytes. In neither case was the HO-2 gene inducible. The expression of the two HO genes led to enzymatic activity in both types of skin cells with an approximately 2.5-fold higher level of enzymatic activity present in keratinocytes compared with fibroblasts derived from the same biopsy. In addition, ferritin levels, which have been found to be augmented via the HO-dependent release of iron from endogenous heme sources, were two- to three-fold higher in keratinocytes compared with matching fibroblasts. This higher ferritin pool would result in an enhancement of cellular iron sequestering capacity that may confer increased resistance to oxidative stress. Indeed, keratinocytes showed less UVA radiation-dependent cell membrane damage than fibroblasts. These results are consistent with the hypothesis that HO expression in human epidermis and dermis is related to cellular defense mechanisms that operate in human skin.

Cells, Cultured↗
PubMed1994

Heme oxygenase 1 mediates an adaptive response to oxidative stress in human skin fibroblasts.

Oxidative stress of human skin fibroblasts by treatment with ultraviolet A (UVA) radiation has been shown to lead to an increase in levels of the heme catabolizing enzyme heme oxygenase 1 [heme, hydrogen-donor:oxygen oxidoreductase (alpha-methene-oxidizing, hydroxylating), EC 1.14.99.3] and the iron storage protein ferritin. Here we show that human skin fibroblasts, preirradiated with UVA, sustain less membrane damage during a subsequent exposure to UVA radiation than cells that had not been preirradiated. Pretreating cells with heme oxygenase 1 antisense oligonucleotide inhibited the irradiation-dependent induction of both the heme oxygenase I enzyme and ferritin and abolished the protective effect of preirradiation. Inhibition of the UVA preirradiation-dependent increase in ferritin, but not heme oxygenase, with desferrioxamine also abolished the protection. This identifies heme oxygenase 1 as a crucial enzymatic intermediate in an oxidant stress-inducible antioxidant defense mechanism, involving ferritin, in human skin fibroblasts.

Adaptation, Physiological↗
PubMed1994

Cellular sensitivity to oxidative stress in the photosensitivity dermatitis/actinic reticuloid syndrome.

Skin fibroblasts from certain patients with the photosensitivity dermatitis/actinic reticuloid syndrome show enhanced sensitivity to ultraviolet radiation compared to normal fibroblasts. To probe further the link between oxidative damage and this disease, we have obtained a more extensive set of cell lines from patients with a severe form of the disease and examined their sensitivity towards oxidative stress by measuring cell survival following UVA radiation (330-450 nm) or hydrogen peroxide treatment (0.1-2.4 mM). The activation of the stress gene, heme oxygenase, has also been assessed by measuring the accumulation of mRNA after hydrogen peroxide treatment. Our studies have confirmed that a slight ultraviolet sensitivity is a characteristic of photosensitivity dermatitis/actinic reticuloid syndrome cell strains and we further demonstrate that these cell lines are particularly sensitive to hydrogen peroxide with up to a three- to fourfold increased sensitivity as compared to normal controls. We also show that certain ataxia telangiectasia strains that are especially sensitive to hydrogen peroxide are also slightly sensitive to ultraviolet radiation. Hydrogen peroxide induces accumulation of mRNA for the oxidant-inducible stress protein, heme oxygenase, with similar kinetics (maximum mRNA accumulation 2-4 h following treatment) and with a similar range of magnitudes in both normal (6.6-20.6 times mRNA increase over basal levels) and photosensitivity dermatitis/actinic reticuloid (2.9-12.8 times) skin cells. Because cells from photosensitivity dermatitis/actinic reticuloid patients show increased sensitivity towards oxidative stress but show no significant change in oxidant activation of the heme oxygenase gene, we propose that the defect involves a late stage of processing of oxidative damage rather than a compromised free radical scavenging system.

Adolescent↗
PubMed1993

Singlet oxygen: a primary effector in the ultraviolet A/near-visible light induction of the human heme oxygenase gene.

Both singlet oxygen and the hydroxyl radical are generated in mammalian cells by UVA (320-380 nm) and possibly near-visible (380-420 nm) radiation. We have modulated the cellular levels of these two reactive oxygen species in order to compare their involvement in the induction of the human heme oxygenase (HO) gene by broad spectrum UVA/near-visible light (UVA/NVL). Irradiation in deuterium oxide (in which singlet oxygen has a longer half-life) enhances the broad spectrum UVA/NVL induction of this gene. Sodium azide and L-histidine which are scavengers of both singlet oxygen and the hydroxyl radical reduce the fluence-dependent accumulation of HO mRNA, while compounds which are only hydroxyl radical scavengers, namely, mannitol and dimethyl sulfoxide do not. Rose Bengal, a known generator of singlet oxygen, also increases the HO mRNA levels, and this induction is enhanced in deuterium oxide. We conclude that the observed effects of deuterium oxide and singlet oxygen scavengers on HO mRNA levels are not due to a nonspecific effect on transcription but that singlet oxygen is a primary effector in the UVA/NVL induction of the human heme oxygenase gene.

Azides↗
PubMed1993

Oxidative stress resulting from ultraviolet A irradiation of human skin fibroblasts leads to a heme oxygenase-dependent increase in ferritin.

Heme oxygenase-1 mRNA levels increase following exposure of many mammalian cell lines to oxidative stress such as ultraviolet A (UVA) irradiation. Here we demonstrate a 4-fold increase in microsomal heme oxygenase activity and a 40% decrease in microsomal heme content 14 h after treatment of human skin fibroblasts (FEK4) with 250 kJ m-2 of UVA radiation. Paralleling this was a 2-fold increase in ferritin levels that was sustained for at least 46 h after UVA irradiation. Treatment of fibroblasts with the iron chelating agent desferrioxamine, after the UVA-dependent induction of heme oxygenase, prevented the increase in ferritin levels. Treatment of fibroblasts with Sn-protoporphyrin IX (an inhibitor of heme oxygenase) also prevented the effect of UVA radiation on ferritin levels. Thus we conclude that the effect of UVA radiation on ferritin levels is via the heme oxygenase-dependent release of iron from endogenous heme sources. We propose that the increase in ferritin that follows UVA irradiation would decrease intracellular free iron such that iron-catalyzed free radical reactions would be restricted during periods of subsequent oxidative stress.

Cells, Cultured↗
PubMed1993

Ultraviolet A (320-380 nm) radiation causes an alteration in the binding of a specific protein/protein complex to a short region of the promoter of the human heme oxygenase 1 gene.

Ultraviolet A (320-380 nm) radiation strongly stimulates expression of the human heme oxygenase 1 gene as a consequence of an enhancement in transcription rate (1). We have used a 147 bp fragment of the promoter of this gene as a probe for DNA binding activity in nuclear extracts prepared from untreated and UVA treated populations of cultured human skin fibroblasts. Analysis using gel electrophoresis mobility shift assays clearly demonstrates the appearance of a strong binding activity unique to UVA-treated extracts that is formed in the absence of de novo protein synthesis. Footprint analysis defines a binding region from -41 to -50 bp that partially overlaps with a region known to constitutively bind upstream stimulatory factor (USF). Further analysis using synthetic oligonucleotides and gel retardation has confirmed that the crucial sequence for binding the protein present in both control and UVA-treated extracts lies within a 26 bp sequence that includes the core USF binding site. UVA radiation appears to lead to a modification of the USF complex (or closely related proteins(s)) to give a structurally modified protein/protein complex which protects only the upstream half of a region originally defined by foot-printing of the promoter with USF.

Base Sequence↗
PubMed1993

The proximal promoter region of the human heme oxygenase gene contains elements involved in stimulation of transcriptional activity by a variety of agents including oxidants.

The rate of transcription of the heme oxygenase gene is enhanced by a variety of agents including oxidants such as hydrogen peroxide and UVA (320-380 nm) radiation and the sulfhydryl reagent, sodium arsenite. To further analyze the inducible response, we have isolated genomic clones of the human heme oxygenase gene. A 1.44 kb fragment corresponding to a region extending from 1416 bp upstream of the mRNA cap site to 24 bp into the 5' untranslated region of the mRNA has been further subcloned and sequenced and used as the basis for the construction of recombinant CAT transient expression vectors. By deleting large portions of this fragment, we have established that elements within 121 bp of sequence immediately upstream of the mRNA cap site respond to various agents (sodium arsenite, hydrogen peroxide, hemin, cadmium chloride and 12-O-tetradecanoyl-phorbol-13-acetate) to give a 3- to 5-fold enhancement in transient expression of the reporter gene. Under the assay conditions employed, induction can only be detected when a SV40 enhancer element is present upstream of the promoter sequence. However, control experiments show that the SV40 sequences serve to amplify the response and are not directly involved in the induction itself. Only a small induction occurs when the entire 1.44 kb fragment is present. The results are consistent with the possibility that additional inducible enhancer elements lie outside of the sequence under study and that a silencer or negative regulatory element occurs upstream of the mRNA cap site within the 1.44 kb fragment.

Base Sequence↗
PubMed1992

Dark induction of haem oxygenase messenger RNA by haematoporphyrin derivative and zinc phthalocyanine; agents for photodynamic therapy.

Haematoporphyrin derivative is one of the main drugs currently used in clinical trials involving photodynamic therapy of cancer, and zinc phthalocyanine is being considered as one of several possible alternatives. We show that incubation of cultured human fibroblasts populations with either of the two drugs will lead to a sharp increase in the accumulation of the messenger RNA corresponding to haem oxygenase. Only cells incubated with haematoporphyrin derivative show additional enhancement of expression of this specific gene on exposure to red light. Since haem oxygenase induction appears to be a specific stress response that may be involved in cellular defence, such observations should be confirmed under conditions which would allow the clinical implications to be fully evaluated.

Cell Line↗
PubMed1992

Endogenous glutathione levels modulate both constitutive and UVA radiation/hydrogen peroxide inducible expression of the human heme oxygenase gene.

Induction of the expression of the mammalian heme oxygenase gene appears to be a general response to oxidant stress. In view of the role of glutathione in protecting cells against solar UVA radiation and other forms of oxidant stress, we have investigated the relationship between intracellular glutathione levels and the inducibility of the human heme oxygenase gene after treatment of populations of cultured skin fibroblasts with either UVA radiation or hydrogen peroxide. We observe a clear relationship between cellular glutathione status and both the constitutive and oxidant-inducible accumulation of heme oxygenase mRNA. Glutathione depletion may lead to enhanced gene expression either as a result of the potentiated accumulation of active oxygen intermediates or as a result of the direct influence of glutathione on a critical target involved in signal transduction.

Antimetabolites↗
PubMed1992

Endogenous glutathione levels modulate the frequency of both spontaneous and long wavelength ultraviolet induced mutations in human cells.

Spontaneous and induced mutations at the hypoxanthine guanine phosphoribosyl transferase locus have been measured in cultured human lymphoblastoid (TK6) cell populations under conditions in which cellular glutathione has been severely depleted by overnight treatment with buthionine-S,R-sulfoximine. At maximum levels of glutathione depletion, the increase in spontaneous frequency is at least 5-fold, a finding consistent with the possibility that cellular redox state can modulate the levels of pre-mutagenic damage arising as a result of normal metabolism in cultured human cells. Glutathione depletion does not lead to a significant enhancement in the frequency of mutants that arise as a result of irradiation at 313 nm but does lead to a 3-fold increase in mutations resulting from irradiation at 365 nm. These results indicate that glutathione may quench reactive intermediates that would otherwise lead to spontaneous mutations as well as a fraction of UVA radiation-induced premutagenic damage.

Buthionine Sulfoximine↗
PubMed1991

Induction of heme oxygenase: a general response to oxidant stress in cultured mammalian cells.

Accumulation of heme oxygenase mRNA is strongly stimulated by treatment of cultured human skin fibroblasts with ultraviolet radiation, hydrogen peroxide, or the sulfhydryl reagent sodium arsenite (S. M. Keyse and R. M. Tyrrell. Proc. Natl. Acad. Sci. USA, 86: 99-103, 1989). Since this will result in a transient reduction in the prooxidant state of cells, the phenomenon may represent an important inducible antioxidant defense mechanism. To examine the generality of the response, we have measured the accumulation of the specific mRNA in a variety of human and mammalian cell types after inducing treatments. Induction by sodium arsenite is observed in all additional human cell types tested. This includes primary epidermal keratinocytes and lung and colon fibroblasts as well as established cell lines such as HeLa, TK6 lymphoblastoid, and transformed fetal keratinocytes. Strong induction of heme oxygenase mRNA is also observed following sodium arsenite treatment of cell lines of rat, hamster, mouse, monkey, and marsupial origin. The agents which lead to induction in cultured human skin fibroblasts fall into two categories: (a) those which are oxidants or can generate active intermediates (ultraviolet A radiation, hydrogen peroxide, menadione, and the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate); (b) agents which are known to interact with or modify cellular glutathione levels (buthionine sulfoximine, sodium arsenite, iodoacetamide, diamide, and cadmium chloride). These observations strongly support the hypothesis that induction of the enzyme is a general response to oxidant stress in mammalian cells and are consistent with the possibility that the cellular redox state plays a key role.

Animals↗