TRANSGENIC ANIMALS FOR THE STUDY OF BIOLOGICAL, PHYSICAL AND CHEMICAL TOXIC AGENTS

Abstract: The invention provides non-human transgenic animals bearing regulatory DNA sequences in some or all their cells, which are sensitive to biological, physical and chemical toxic agents. Such sequences are linked to sequences of reporter genes useful for toxicological studies.

Description: TRANSGENIC ANIMALS FOR THE STUDY OF BIOLOGICAL, PHYSICAL AND CHEMICAL TOXIC AGENTS The present invention provides transgemc animals for the study of biological, physical and chemical toxic agents . At present, toxicity tests can be carried out both in vivo and m vitro. The industrials, the public opinion and the scientific community are strongly interested m the abolition of toxicity tests made on animals and therefore in their replacement with in vitro tests. This target, however, is quite unrealistic at the moment, since no in vitro tests which can replace in vivo tests are available, either now or m the near future . It is well known, in fact, that the substances under in vivo investigation often undergo metabolic modifications, which might significantly alter their toxicity profile, to an extent which would be unpredictable m in vitro tests. On the other hand, m vivo studies always involve animal suffering and sacrifice. However, it is possible to conceive genetically- engineered animal models which may simplify the determination of the toxicity of various agents and reduce the number of animals involved. Recently, the use of transgemc animals as models for pharmacological studies has been proposed. For example, EP 0 169 672 Bl describes transgemc animals bearing oncogeneε like c-myc, suitable for the study of tumors associated to the expression of such oncogenes, or bearing the human growth hormone gene fused to a metallothionein promoter, whereby, said promoter being an inducible promoter, it is possible to study the effect of the expression, upon induction, of the associated gene on the whole organism (Palmiter et al. (1983) Science 222, 809). WO 91/15579 describes a method for studying mutage- neεis in transgenic animals bearing DNA sequences which can easily be extracted and analysed for mutations. The present invention provides non-human transgenic animals useful for toxicity studies. Such animals are characterised in that they have regulatory DNA sequences in some or all their cells, which are sensitive to biological, physical and chemical toxic agents, functionally linked to sequences of reporter genes, whereby the expression of the latter sequences is controlled or induced by said regulatory sequences . Among the regulatory sequences, the stress-promoter sequences, like the heat shock protein (hεp) promoters, are preferred, but also cytochrome-promoters of the p450-superfa ily , as well as those promoters of other genes, like p53 gene, activated by biological, chemical or physical stress, can be cited. Among suitable reporter genes, the growth hormone gene, which has been used in the experiments described below, is preferred, but also chlorarnphenicol acetyl tranεferase (CAT), green fluorescence protein (GFP) and β-galactosidaεe ( acZ) genes can be suitably employed. The transgenic animals of the invention can be used m a method for studying the toxicity induced by various agents. In theory, any animal normally suitable for a toxicity test can be used the method of the invention In practice, non-human mammals, particularly primates and rodents, are preferred. Mice, in particular, are the most preferred. Conventional ethodε can be uεed for the production of transgemc animals, including, for example, the micromiection of recombmant DNA into embryonal cells or into pronuclei of one-cell stage embryos, the zygote , embryo cell, somatic cell or animal tisεue infection with a virus, m particular with a retrovirus, according to what described, for example, in Hogan et al., Cold Spring Harbor Laboratory Press, NY, 1986; Pal iter et al., Ann. Rev. Genet., 20: 465-499; 1986; Capecchi, Science, 244: 288-292, 1989. The method for the m vivo assay of potential toxic compounds according to the present invention, compriseε exposing the animal to a chemical or phyεical agent for a time sufficient to induce the effect, and simply measuring the reporter gene expression. When the reporter gene encodes a protein secreted in the bloodstream, for instance, its hematic concentration, as well as other chemical-clinical parameters associated with the effect caused by the activation of the stress promoter, could be detected. According to the first aspect of the invention, a preferred embodiment is the production of transgenic mice in which a construct has been inserted, which comprises a hsp promoter fused to growth hormone (GH) gene (transgene), said promoter being described Dreano et al. (Biotechnology u6:953, 1988 and Gene 49:1- 8, 1986) and in Fishbach et al . (Cell Biol . Toxicol . 9:177-188, 1993). The latter publication reports that the exposure to toxic metals of a stable fibroblast line, engineered with a construct containing the growth hormone gene under the control of hsp promoter, causes the secretion of the reporter gene in the medium. According to the preferred embodiment of the invention, the injury caused by the toxic agent is determined as the increase of GH plasma concentration versus the control. This model has resulted particularly efficient and sensitive, especially in relation with toxic metals, but it can suitably be used also for other classes of chemical toxic compounds, like endocrine diεruptorε, as well as for other physical or chemical agents, like radiations and electromagnetic fields. The ma advantages offered by the invention are: the possibility to diminish animal suffering, since only low amounts of the teεt εubεtances are used, surely lower than the dosages which could induce animal suffering or death; the reduction of the number of animals used m toxicological tests; the provision of a model that is absolutely reliable for what concerns the metabolic modifications, which the toxic agents undergo in the organism, the interactions of toxic compounds with various organs and their final effects on cells, including the chronic effects. This model is particularly useful for teεt reiterations and allows to monitor the agent's effect during long-lasting treatments using always the εame animal, thuε eliminating the variability of the individual reεponεe. Further, εeveral compounds can be studied using the same animal. Finally, such transgenic models can be used also for in vivo studies of toxicity kinetics of toxic compounds . The second aspect of the invention concerns the possibility to obtain primary cultures of cells from different tiεsueε of the transgenic animal, in which a recombmant DNA construct is integrated as described above, whereby a cell- or tiεεue-εpecific toxicity study can be carried out and the intracellular biochemical effects connected to toxicity can be evaluated under controlled conditions and in more detail during different stages of animal growth. In this case, the in vitro aεsay compriεes preparing primary cultures in conditions variable depending on the cell type, exposing said cultures to the toxic agent and monitoring the activation of the stress promoter through detection of the protein encoded by the reporter gene. Referring to the above described transgenic mice bearing the hsp/GH construct, an embodiment of the second aspect of the invention consists for example in preparing primary cultures of fibroblasts, kidney, lung or bone marrow cells, hepatocytes or other, in their εimultaneouε or εeparate treatment with one or more toxic agentε, and in the determination of GH secretion in the medium. If, using the above assay, a tisεue or a cell-type resulted sensitive to the toxic agent, a deeper biochemical analysis could be made m order to find which cellular pathways are particularly involved m the toxicity . Thuε, according to a further aspect, the invention provides a method to carry out vitro toxicity tests on primary cultures of somatic cells derived from a transgemc animal . BRIEF DECRIPTION OF THE FIGURES Fig 1. Panel A: Southern blot analysis of transgenic heterozygous (lanes 1-4) and ho ozygous mice (lanes 5-7) and a non-transgemc control mouse (lane 8). Panel B: RT-PCR with hGH specific primers of heat- shock activated liver cells from transgenic mice. Samples: RNA from cultured hepatocyteε before (lane 1) and 30 mm after (lane 2) heat εhock in vitro; RNA from livers before (lane 3) and 30, 60, 90, minutes after heat εhock (lanes 4-6). + and - represent the negative and positive controls reεpectively . Lanes 7 to 10 are the amplifications on non-retrotranscribed liver RNAs performed on the same samples as in lanes 3 to 6. Ml: marker V, M2: 1 kb ladder. Panel C: RT-PCR with HPRT specific primers performed on RNAs from the sampleε 1 to 6 aε m panel B. Fig. 2: Plasma levels of hGH (pg/ml) measured at different times in transgenic mice after thermal εtreεε. Values represent the mean ± SE; the number of mice tested for each time period is indicated by the number above each bar. Fig. 3: Mean hGH plasma levels (pg/ml) ± SE observed transgenic mice injected l.p with PBS and with various inorganic toxic compounds at the indicated doses. Besides controls, are indicated: Rb : rubidium chloride; Hg : methylmercurium chloride; Cu copper sulphate; Cd : cadmium chloride; As- sodium arεemte (2 doεes) (below each bar is given the number of tested mice). The levels of significance are: p<0.05; **p<0.01; ***p<0.005 Fig. 4: Mean ± SE of plasma hGH levelε observed in transgenic mice subjected to two consecutive treatments, according to the following schema: Group First Second Time treatment treatment Interval (T χ ) (T 2 ) (τ 1 -τ 2 ) As^ As Aε 10 days Aε 2 Cd As 2 months Aε 3 Rb As 2 months Cu Cu Cu 2 months Control untretated untreated The following examples better llluεtrate the invention: EXAMPLE 1 Production and characterization of a transαemc mouse lineage Transgenic mice were produced according to standard techniques (Hogan et al., "Manipulating the mouse embryo: a laboratory manual", Cold Spring Harbor Laboratory Presε, Cold Spring Harbor, N.Y., 1986), by microinjectmg 1-cell εtage embryo pronuclei with a 1.4 kb EcoRI DNA fragment from pl7hGH conεtruct (described m Dreano et al., Biotechnology 6:953, 1988 and Gene 49:1-8, 1986), containing the human growth hormone cDNA aε reporter gene, fused to the control region of the human Hεp70 promoter. Mice were screened by Southern blot and/or PCR performed on tail DNA according to standard techniques. PCR was performed with the following primers: hGH : GTGCAGTTCCTCAGGAGTGT; hGHR : CGAACTTGCTGTAGGTCTGC . The amplification product was 171 bp long. Amplification conditions (35 cycles) were: 94°C for 20 sec, 58°C for 30 sec and 72°C for 20 εec . Heterozygous males and females were crossed and the homozygous progeny was identified by Southern blot, based on the intensity of the transgenic bands; their homozygoεity waε confirmed by checking the offspring when the homozygous male was mated to a non-transgenic partner. The mice uεed for the in vitro and in vivo experimentε were always derived from a homozygouε male bred with a non-tranεgenic CD-I female. Total RNA waε extracted from different tiεεueε (liver, εpleen, lung, kidney, blood) of tranεgenic and control mice, according to standard techniques ( Sambrook et al., "Molecular cloning: a laboratory manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Southern and Northern blot were performed according to standard techniqueε. In order to evaluate the baεal value of non-induced expreεεion of the tranεgene, mice were analysed with Northern blot and with RT-PCR. No expreεεion waε detected in lung, kidney, εpleen, liver and peripheral blood lymphocytes of non-treated animals or of animals not-exposed to heat shock. The hGH level in non-treated mice (control) waε generally under the test detection limits, and when it was determined, it never exceeded 10 pg/ml. EXAMPLE 2 Tn vivo heat shock treatment. Eight transgenic mice obtained according to example 1 and four non-transgenic control mice were subjected to in vivo heat shock at 44"C for 30 in. Six additional unexpoεed transgenic mice were teεted. Aliquots of blood were taken before and 1, 3, 5, 7, and 24 hours after the heat shock. In transgenic mice (Fig. 2) a specific increase of plasma hGH was detected with a peak three hour after treatment . These resultε suggest that the integrated transgene doeε not affect in vivo the normal reεponsiveneεε of hsp promoter . EXAMPLE 3 a) Inducibility of the hsp70/hGH tranεgene expreεsion in vivo by εodiu arεenite and methylmercurium chloride. Male tranεgenic mice obtained as described in example 1 were weighed, anesthetized with ether and injected intraperitoneally (i.p.) with NaAεO^ diεεolved in PBS, at a final dose of 2.5 or 5 mg/kg, or with 3.5 mg/kg CH^HgCl disεolved in PBS. Control tranεgenic mice were injected with the same volume of PBS (about 200 μl/mouse ) . Blood εamples were recovered before injection and 1, 3, 5, 7 and 24 hours after treatment. hGH plasma levels at different times and doses are shown in Fig. 3. Both the teεted doεeε of NaAε0 2 gave a clear and statistically significant response The responεe peaked after 3-5 hours and turned to the basal level 24 hours after injection CH HgCl gave hGH peaks after 5-7 hours and baseline hGH valueε 24 hours after injection. b) Following the same procedure aε deεcribed m a), hGH inducibility waε evaluated m mice treated with rubidium chloride (18.5 mg/kg, c), copper εulfate (9 mg/kg, d) and cadmium chloride (4.7 mg/kg, e). Results are reported in Fig. 3. XAMPL 4 Inducibility of the hsp70/hGH tranεαene expression in vivo by repeated lmections of toxic compounds . Initially, 13 mice were treated aε follows: 5 mice with As, 3 mice with Cd, 2 mice with Rb, 3 mice with Cu . After a period of 10 dayε to 2 months, the former three groupε of mice were re-moculated with Aε , the latter with Cu. Blood samples were taken before and 3-5 hours after injection, I e . at the timeε of higheεt reεponεe. As shown m Fig. 4, after the first administration of the compound, the mice showed a response comparable to that observed in groups of mice treated as m example 3 When retested after 10-60 dayε, a similar hGH increase was observed. EXAMPLE 5 Embryonic fibroblast primary cultures-in vitro toxicity tests. Homozygous transgenic mice obtained aε described in example 1 were croεεed with CD-I femaleε After 14 days, embryonic fibroblasts (EMFIS) were recovered from the fetuεeε according to the technique deεcribed by Robertson E.J., IRL Press, Oxford, 77-88, 1987. Cellε were cultured in DMEM supplemented with 10% FCS and antibiotics (pen/strep), in an incubator (C0 2 :5%, 100% humidity). Culture medium waε replaced every second day with pre-warmed (37°C) fresh culture medium. The cells were expanded for two passages and then frozen at -80°C. For each experiment, cells were thawed, plated in 10 cm Petri dishes, left to grow and then re-seeded on 12 well plates until confluence. To evaluate the toxic effect of the compounds, cells were treated by substituting the culture medium with fresh pre-warmed serum-free medium containing the toxic compounds at the chosen final dilutions. Cells were exposed to the toxic compound for either 5 or 24 hours and then the medium waε replaced with fresh control medium for an additional 24 hours. At the end of the treatment, culture media were collected and asεayed for hGH secretion by enzyme immunoassay. Each treatment was performed in triplicate and the hGH determination waε repeated twice for each plate. The results are expresεed as pg of hGH/10^ cells. The sensitivity of this method waε approximately 2-4 pg/ml. As shown m the table, calcium and rubidium, known for their lack of toxicity at the tested concentrations, do not provoke hGH release the medium. On the contrary, a significant release is induced after 24 hours of chrome exposure, while copper gives a low reεponεe after 24 hourε at the higheεt concentrations. On the contrary, mercurium doeε not induce hGH release from fibroblasts at each tested concentration. Finally, arsenic and cadmium, as expected, showed clearly toxic. EXAMPLE 6 Primary hepatocvtes cultureε-in vitro toxicitv tests. Tranεgenic male mice 8 weekε old were anesthetized and their livers were perfuεed aε deεcribed in Clerici et al., Mut. Res., 227:47-51, 1989, in order to collect hepatocytes. Hepatocyteε were then εeeded on 24 well plateε (2x10^ cellε/well) and cultured in William'ε E medium supplemented with antibiotics (pen/strep) and 10% FCS for 2 hours in order to allow them to attach to the bottom of the Petri disheε. The supernatant was then removed and the adherent cellε were treated with the compoundε diεsolved in the medium. To evaluate the toxic effect of the compoundε, cells were treated by subεtituting the culture medium with fresh pre-warmed serum-free medium containing the toxic compounds at the chosen final dilutionε. Aε εhown in the table, calcium and rubidium do not induce hGH releaεe by mature hepatocyteε. Chrome treatment induceε a high response after 24 hours, while copper treatment causes releaεe either after 5 or 24 hourε at each concentration. Mercurium induces a response at concentrations higher than 5xl0-^ M, while arεenic and cadmium εhow extremely toxic. EXAMPLE 7 In vitro toxicity testε on kidney, lung and bone- marrow primary cultures. Kidney and lung cellε were recovered as deεcribed by Campbell, J. A. et al. ("Siεter cromatid exchange analysis of mice following in vitro exposure to vinyl carbonate", In vitro Cell. Dev . Biol. 22: 443:448, 1986) . Briefly, kidneys were removed from the same animals subjected to liver perfuεion, waεhed 3 timeε in PBS additioned with antibioticε and minced in 0.5 mm pieceε with a εterile εcalpel. After 1 hour of incubation in trypεin/collagenaεe (lOOU/ml) εolution, the suεpenεion waε centrifuged twice for 5 min. at 50xg, plated in 100 mm Falcon disheε and cultured in McCoy 'ε medium with 20% FCS, 2mM Glutamine and Pen/εtrep. In order to collect lung cellε, after liver perfusion the chest cavity was opened after liver perfuεion to acceεε the lungε. The trachea waε cut with a scalpel and a 22-gauge catheter waε inserted into the trachea to perfuse the lungs with trypsin/collagenase εolution for 5 min. in order to help the disaggregation of this tissue. The cells were then trypsinized, εeeded in 24 wells and left to grow until confluence in McCoy's medium with 20% FCS, 2mM Glutamine and antibiotics. In order to prepare bone marrow primary cultures, bone marrow cells were flushexd from the cavity of femurs and tibias with a εyringe containing the culture medium. Cellε were plated in 12 well plateε with McCoy' ε medium with 20% FCS, 2mM Glutamine and antibioticε, and left to grow until the εtromal cellε reached confluence. To evaluate the toxic effect of the compoundε, the same procedure waε followed aε in the above examples 5 and 6. Results are reported in the Table. Table (A) Determination of hGH (pg/10" cells) release and primary transgenic cultures viability after 5-hour treatment hGH re lease Viabi •lty , Compounds Primary lines 10 -5 M 5x10 " -5 M 10 "4 M 5xl0 _4 M 10 _5 M 5x10 " °M 10 _4 M 5xlO _4 M CaCl 2 hepatocytes nd nd nd nd + + + + RbCl nd nd nd nd + + + + CrCl 3 / nd nd nd / + + + CuS0 4 / nd 80 66 / + + + K 2 Cr20 7 nd 65 94 65 + /- + /- - - CH 3 HgCl nd nd nd / + /- +/- - / CdCl 2 309 452 57 14 + /- +/- - CJ1 NaAs0 2 100 224 nd / + +/- - / CaCl 2 E bryonic / nd nd nd / + + + RbCl f ibroblast / nd nd nd / + + + CrCl 3 / nd nd nd / + + + CuS0 4 / nd 6 12 / + + + K 2 Cr20 7 9 nd nd nd + /- + /- - - CH 3 HgCl / nd nd nd / + /- - / CdCl 2 250 85 45 nd + /- +/- - - NaAs0 2 nd 113 19 nd + + /- + /- / cont inues CaCl 2 Kidney / nd nd nd / + + + RbCl cells / 57 nd nd / + + + CrCl 3 / nd 15 nd / + + +/ CuS0 4 / nd nd nd / + +/- +/ KnCr n J- j nd nd nd / +/- +/- +/- / CH 3 HgCl 10 nd nd / +/- +/- +/- / CdCl 2 nd nd nd / +/- - / NaAsO 22 17 28 / + +/- - / CaCl 2 Lungs / nd 127 202 / + + + RbCl cells / 28 191 71 / + + + CrCl 3 / 92 122 166 / + + + CTl CuS0 4 / nd nd 184 / +/- +/- +/ K 2 Cr 2 0 7 nd nd nd / +/- - / CH 3 HgCl 27 nd nd / - / CdCl 2 nd 31 11 / +/- - / NaAsOo nd 37 249 / +/- + / ■ - / hGH levels in untreated cells medium (controls) were not measurable after 5-24-hour incubation . nd = undetectable; / = not determined; + = with 100% viability; with 30-70% viability; - = 100% dead (R) Determination of hGH (pg/10^ cells) release and primary transgenic coltures after 24- hour treatment. hGH release Vitality λ Compounds Primary lines 10 _5 M 5x10 " "5 M 10 ~4 M 5xlO ~4 M 10 _5 M 5x10 " " ^M 10 _4 M 5xl0 -4 M CaCl 2 hepatocytes nd nd nd nd + + + + RbCl nd nd nd nd + + + + CrCl 3 / 36 20 nd / + + - CuS0 4 / 12 61 100 / + + + /- K 2 Cr 2°7 nd nd nd nd - - - - CH 3 HgCl nd 63 103 / + /- - - / CdCl 2 nd nd 17 21 + /- + /- - - NaAs0 2 270 19 5 / + + /- - / CaCl 2 Embryonic / nd nd nd / + + + RbCl f ibroblast / nd nd nd / + + + continues CaCl 2 Kidney / nd nd nd / + + + RbCl cells / nd nd nd / + + + CaCl 2 Lungε / nd nd nd / + + + /- RbCl cells / 20 110 114 / + + + /- CaCl 2 Bone marrow / nd 51 nd / + + + /- RbCl cells / nd 20 128 / + + + /- hGH levels in untreated cells medium (controls) were not measurable after 24-hour incubation. nd = undetectable; / = not determined; + = with 100% viability; with 30-70% viability; - = 100% dead

Claims: CLAIMS 1. A non-human tranεgenic animal which comprises cellε containing a construct of a stress-εenεitive regulatory sequence linked to a reporter-gene sequence. 2. A non-human tranεgenic animal according to claim 1, wherein εaid regulatory εequence iε the heat shock protein (hsp) promoter. 3. A non-human transgenic animal according to claim 2, wherein said εequence is hsp70 gene promoter. 4. A non-human transgenic animal according to claims 1-3, wherein said reporter gene is the growth hormone (GH) gene. 5. A non-human transgenic animal according to any of the previous claims, which is a mammal. 6. A non-human transgenic animal according to claim 5, which is a rodent. 7. A non-human transgenic animal according to claim 6, which iε a mouse. 8. A primary cell culture obtained from the transgenic animals of claims 1-7, wherein cells bear a construct of a stress-sensitive regulatory sequence linked to a reporter-gene sequence. 9. A primary cell culture according to claim 8, which is a fibroblaεt, hepatocyte, kidney, lung and bone marrow-cell culture. 10. A method for the study of chemical, physical and biological toxic agents which comprises: a) exposing the transgenic animal of claims 1-7 to the toxic agent; b) determining the effect through measurement of the reporter-gene expreεεion. 11. A method according to claim 10, wherein the εame animal iε uεed for repeated teεtε with the εame or different toxic agent. 12. A method according to claimε 10-11, for the εtudy of toxicity kinetics of one or more toxic agents. 13. A method according to claims 10-12, for the study of heat stress. 14. A method according to claims 10-12, for the study of metal toxicity. 15. A method according to claim 14 for the study of toxicity of metals selected from the group consisting of Rb, Cu, Hg, As and C . 16. A method for the toxicity study of chemical, physical and biological agents, which comprises: a) preparing a primary culture from the tranεgenic animal of claims 1-7, in which the cultured cells bear a construct of a streεε- εensitive regulatory sequence linked to a reporter-gene sequence; b) exposing the primary culture to the toxic agent; c) determining the effect through the expression of the reporter gene in the culture medium. 17. A method according to claim 16, wherein fibroblaεt and hepatocyte primary cultureε are uεed. 18. A method according to claimε 16-17 for the εtudy of metal toxicity. 19. A method according to claim 18, wherein metalε are εelected from the group conεiεting of Rb, Cr, Cu, Hg, Aε , and Cd . 20. The uεe of the tranεgenic animal of claim 1 for in vivo toxicity εtudies. 21. The uεe of a tranεgenic animal according to claim 19, wherein εaid animal iε a mouεe. 22. The uεe of primary cultureε of cellε from the tranεgenic animal of claim 1, for in vitro toxicity studies .

Inventors: BROMLEY PETER, CLERICI LIBERO A, RONCUCCI ROMEO DI, SACCO MARIA GRAZIA, VEZZONI PAOLO, ZECCA LUIGI, BROMLEY, PETER, CLERICI, LIBERO, A., RONCUCCI, ROMEO +DI, SACCO, MARIA, GRAZIA, VEZZONI, PAOLO, ZECCA, LUIGI
Applicants: BROMLEY PETER, CLERICI LIBERO A, CONSIGLIO NAZIONALE RICERCHE, SACCO MARIA GRAZIA, VEZZONI PAOLO, ZECCA LUIGI, BROMLEY, PETER, CLERICI, LIBERO, A., CONSIGLIO NAZIONALE DELLE RICERCHE, SACCO, MARIA, GRAZIA, VEZZONI, PAOLO, ZECCA, LUIGI
Application year: 1998
Main classification: C12N15
IPC codes: A01K67, C12N5, C12N15, C12Q1, G01N33